A compound preparation with the efficacy of supplementing qi and promoting blood production, its preparation method and application

Abstract

The present invention provides a compound preparation with the efficacy of supplementing qi and generating blood, its preparation method and application, belonging to the field of pharmaceutical technology. Based on the basic formula of ginseng, rehmannia root, codonopsis pilosula, atractylodes macrocephala, and hawthorn, the method of binary eutectic solvent extraction and ternary eutectic solvent extraction is successively adopted to extract the active ingredients in the basic formula, and then donkey-hide gelatin is added to obtain the compound preparation. The yield of the compound preparation obtained by the present invention is above 43%, and the contents of the important active ingredients polysaccharide, saponin, and flavonoid can reach above 26%, 23%, and 9% respectively. In addition, the compound preparation obtained by the present invention has good biological activity, and has the effects of improving anemia, enhancing immunity, promoting microcirculation, anti-inflammatory, whitening, etc., and can be used as a raw material in the fields of preparing functional foods, health products, and even drugs, etc., and has important economic value and social benefits.

Description

Technical Field

[0001] The present invention relates to the technical field of medicine, and particularly to a compound preparation with the efficacy of supplementing qi and promoting blood production, and its preparation method and application. Background Art

[0002] Life is based on qi and blood, and human health and aging are closely related to qi and blood. The syndrome of qi and blood deficiency is one of the common syndromes in traditional Chinese medicine. Long-term qi and blood deficiency in the body is likely to cause symptoms such as physical fatigue, tiredness, drowsiness, and low immunity, and is also prone to trigger other diseases. In women, qi and blood deficiency can easily lead to anemia. During the treatment of anemia, most Western medicines are related symptomatic treatments for various clinical indications, such as immunoglobulins, thymosin, transfer factor and other drugs for enhancing immunity, and dextran iron, ferrous sulfate, folic acid, vitamin B 12 and other drugs for treating anemia. Compound traditional Chinese medicine preparations are an important part of traditional Chinese medicine in our country. Due to the diversity of their chemical components, they can play a unique pharmacological role in different syndromes and various development stages of sub-healthy people with qi and blood deficiency. Based on referring to the century-old ancient prescription "Liangyi Ointment" in "Complete Works of Jingyue", modern medical experts have screened out a new formula combination based on six Chinese medicinal materials including ginseng, prepared rehmannia root, combined with donkey-hide gelatin, codonopsis pilosula, atractylodes macrocephala, and hawthorn, which has good application value in exerting the efficacy of supplementing qi and promoting blood production.

[0003] Through long-term practice and accumulation, ancient doctors realized that the extraction methods of traditional Chinese medicine also need to follow certain rules. Xu Lingtai, a medical expert in the Qing Dynasty, pointed out that "the method of decocting medicine should be deeply studied, and the effectiveness of the medicine depends entirely on this". Modern research shows that the extraction method of traditional Chinese medicine is important because the chemical components will change in quantity and quality during the extraction process, which will lead to changes in the efficacy of the medicine. For example, research shows that the alcohol extract of astragalus membranaceus can increase the endurance index and adrenal gland index of mice, while the water extract only has the effect of increasing body weight. Traditional extraction methods of traditional Chinese medicine mainly rely on operations such as decocting and soaking. However, with the progress of technology, the current extraction and production process of traditional Chinese medicine is undergoing unprecedented changes. While maintaining traditional advantages, the traditional Chinese medicine industry needs to better meet the needs of modern society, effectively improve the quality and efficiency of traditional Chinese medicine, and achieve the perfect combination of traditional Chinese medicine wisdom and modern technology.

[0004] Some biocompatible hydrogen bond acceptors and hydrogen bond donors can produce strong intermolecular hydrogen bond interactions in a specific ratio to form a deep eutectic solvent. The deep eutectic solvent can have a good extraction effect on the chemical components in some traditional Chinese medicines, and has advantages that cannot be compared with organic solvents such as being green and environmentally friendly. For example, Chinese Patent Document CN112375075A (Application No.: 202011136677.X) discloses a method for extracting puerarin using a natural deep eutectic solvent. The deep eutectic solvent constructed by this method is composed of a hydrogen bond acceptor (such as L-proline) and a hydrogen bond donor (such as malic acid), with a short extraction time, low energy consumption, and realizing the enrichment of puerarin in kudzu root. Chinese Patent Document CN112439000A (Application No.: 201910819706.3) discloses a method for extracting and purifying flavonoids in Ginkgo biloba leaves by combining deep eutectic solvent extraction with macroporous resin. The deep eutectic solvent constructed by this method is composed of choline chloride and polyol. Combining with the macroporous resin adsorption process, the content of flavonoids obtained after purification is higher than 24%, meeting the requirements for the content of flavonoids in the Ginkgo biloba leaf extract in the Chinese Pharmacopoeia (2015 Edition).

[0005] For a new formula compatibility based on six traditional Chinese medicine materials including ginseng, prepared rehmannia root (prepared radix rehmanniae), donkey-hide gelatin, codonopsis pilosula, atractylodes macrocephala, and hawthorn, in order to make it fully exert the efficacy of supplementing qi and generating blood, the present invention intends to construct a new method for extracting traditional Chinese medicine components to achieve efficient extraction of the active ingredients in this formula and make it achieve more excellent active effects in an effective and safe manner. Summary of the Invention

[0006] The present invention provides a compound preparation with the efficacy of supplementing qi and generating blood, its preparation method and application. Based on the basic formula of ginseng, prepared rehmannia root, codonopsis pilosula, atractylodes macrocephala, and hawthorn, the active ingredients therein are extracted successively by binary deep eutectic solvent extraction and ternary deep eutectic solvent extraction techniques, and finally supplemented with donkey-hide gelatin. The operation process is simple and the extraction effect is excellent. The yield of the obtained compound preparation is above 43%, and the contents of polysaccharides, saponins, and flavonoids can reach above 26%, 23%, and 9% respectively, greatly obtaining the high-quality active ingredients of traditional Chinese medicine.

[0007] The technical solution of the present invention is as follows:

[0008] A preparation method of a compound preparation, based on the basic formula of ginseng, prepared rehmannia root, codonopsis pilosula, atractylodes macrocephala, and hawthorn, successively adopts the methods of binary deep eutectic solvent extraction and ternary deep eutectic solvent extraction to extract the active ingredients in the basic formula, and then adds donkey-hide gelatin to obtain the compound preparation;

[0009] Wherein, the binary deep eutectic solvent includes a hydrogen bond acceptor and hydrogen bond donor A, and the ternary deep eutectic solvent includes a hydrogen bond acceptor, hydrogen bond donor A, and hydrogen bond donor B;

[0010] Among them, the hydrogen bond acceptor is one of betaine, aspartic acid, and phenylalanine; the hydrogen bond donor A is one of ethylene glycol, glycerol, 1,4-butanediol, and glucose; the hydrogen bond donor B is one of citric acid, lactic acid, acetic acid, malonic acid, and oxalic acid.

[0011] The preparation method of the compound preparation specifically includes the following steps:

[0012] (1) Prepare the basic formula according to the weight ratio: 5-25 parts of ginseng, 10-30 parts of rehmannia root, 20-35 parts of codonopsis pilosula, 10-30 parts of atractylodes macrocephala, 5-25 parts of hawthorn; dry and crush the medicinal materials and mix them evenly to obtain medicinal material powder. Add a binary eutectic solvent composed of a hydrogen bond acceptor, a hydrogen bond donor A, and water to the medicinal material powder, and perform ultrasonic extraction at 60-80 °C for 0.5-3 h, then filter to obtain extraction solution 1 and medicinal material filter residue;

[0013] (2) Add AB-8 macroporous resin to the extraction solution 1 in step (1), oscillate at 50-130 r / min at room temperature for 8-12 h, take out the macroporous resin to obtain an adsorption solution; desorb the taken-out macroporous resin with 95% (v / v) ethanol to obtain a desorption solution, concentrate and dry the desorption solution to obtain extract 1;

[0014] (3) Add the hydrogen bond donor B to the adsorption solution in step (2) so that the binary eutectic solvent in the adsorption solution forms a ternary eutectic solvent with the hydrogen bond donor B. Use the ternary eutectic solvent to perform ultrasonic extraction on the medicinal material filter residue in step (1) at 60-80 °C for 0.5-3 h, then filter to obtain extraction solution 2; add 4-6 times the volume of absolute ethanol to extraction solution 2, perform alcohol precipitation for 10-14 h, centrifuge, and dry the precipitate after centrifugation to obtain extract 2;

[0015] (4) Take 1-5 parts by weight of donkey-hide gelatin, crush it, and mix it evenly with extracts 1 and 2 to obtain the compound preparation.

[0016] Preferably, in the binary eutectic solvent in step (1), the hydrogen bond acceptor is one of betaine, aspartic acid, and phenylalanine, and the hydrogen bond donor A is one of ethylene glycol, glycerol, 1,4-butanediol, and glucose; the molar ratio among the hydrogen bond acceptor, the hydrogen bond donor A, and water is 1:(0.5-3.5):(1.5-5); the binary eutectic solvent is prepared by mixing the hydrogen bond acceptor, the hydrogen bond donor A, and water, and heating and stirring at 75-90 °C for 0.5-3 h.

[0017] More preferably, in the binary deep eutectic solvent in step (1), the hydrogen bond acceptor is one of aspartic acid and phenylalanine, and the hydrogen bond donor A is one of ethylene glycol and glucose; the molar ratio of the hydrogen bond acceptor, the hydrogen bond donor A and water is 1:(1-3):(2-4.5); the binary deep eutectic solvent is prepared by mixing the hydrogen bond acceptor, the hydrogen bond donor A and water, and heating and stirring at 80-90 °C for 1-2 h.

[0018] Even more preferably, in the binary deep eutectic solvent in step (1), the hydrogen bond acceptor is phenylalanine, and the hydrogen bond donor A is glucose; the molar ratio of the hydrogen bond acceptor, the hydrogen bond donor A and water is 1:2:3; the binary deep eutectic solvent is prepared by mixing the hydrogen bond acceptor, the hydrogen bond donor A and water, and heating and stirring at 85 °C for 1 h.

[0019] Preferably, the ratio of the medicinal material powder to the binary deep eutectic solvent in step (1) is 1:(5-25) (g / mL).

[0020] Preferably, the ratio of the AB-8 macroporous resin to the extract 1 in step (2) is 1:(3-10) (g / mL), and the amount of 95% (v / v) ethanol used is 5-15 times the volume of the AB-8 macroporous resin.

[0021] More preferably, the ratio of the AB-8 macroporous resin to the extract 1 in step (2) is 1:(5-8) (g / mL), and the amount of 95% (v / v) ethanol used is 8-12 times the volume of the AB-8 macroporous resin.

[0022] Even more preferably, the ratio of the AB-8 macroporous resin to the extract 1 in step (2) is 1:6 (g / mL), and the amount of 95% (v / v) ethanol used is 10 times the volume of the AB-8 macroporous resin.

[0023] Preferably, in the ternary deep eutectic solvent in step (3), the hydrogen bond donor B is one of citric acid, lactic acid, acetic acid, malonic acid, and oxalic acid; the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor B is 1:(0.5-3); the ternary deep eutectic solvent is prepared by adding the hydrogen bond donor B to the adsorption solution and heating and stirring at 75-90 °C for 0.5-3 h.

[0024] More preferably, in the ternary deep eutectic solvent in step (3), the hydrogen bond donor B is one of citric acid, lactic acid, and malonic acid; the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor B is 1:(1-2); the ternary deep eutectic solvent is prepared by adding the hydrogen bond donor B to the adsorption solution and heating and stirring at 80-90 °C for 1-2 h.

[0025] More preferably, in the ternary eutectic solvent described in step (3), the hydrogen bond donor B is lactic acid; the molar ratio between the hydrogen bond acceptor and the hydrogen bond donor B is 1:1; the ternary eutectic solvent is prepared by adding the hydrogen bond donor B to the adsorption solution and heating and stirring at 85 °C for 1 h.

[0026] Preferably, the pulverization particle size of the donkey-hide gelatin in step (4) is 500-1500 mesh.

[0027] Further preferably, the pulverization particle size of the donkey-hide gelatin in step (4) is 800-1200 mesh.

[0028] More preferably, the pulverization particle size of the donkey-hide gelatin in step (4) is 1000 mesh.

[0029] A compound preparation prepared by the above preparation method.

[0030] Application of the compound preparation in the preparation of products for treating anemia, products for improving microcirculation, products for enhancing immunity, anti-inflammatory products, and whitening products.

[0031] Beneficial effects:

[0032] (1) In the present invention, a ternary eutectic solvent extraction system is constructed by sequentially selecting suitable hydrogen bond acceptors and hydrogen bond donors, which has good extraction efficiency for various pharmacodynamic components in the basic formula for supplementing qi and promoting blood production composed of ginseng, rehmannia root, codonopsis pilosula, atractylodes macrocephala, and hawthorn. Finally, donkey-hide gelatin is supplemented, and the yield of the obtained compound preparation is above 43%, and the contents of important active components such as polysaccharides, saponins, and flavonoids can reach above 26%, 23%, and 9% respectively.

[0033] (2) The compound preparation obtained in the present invention has good biological activity and has the effects of improving anemia, enhancing immunity, promoting microcirculation, anti-inflammation, whitening, etc., and can be used as a raw material in the fields of preparing functional foods, health products, and even drugs, etc., and has important economic value and social benefits.

[0034] (3) The new extraction process provided by the present invention can better retain the active components of traditional Chinese medicine and improve the extraction efficiency, so as to better meet the needs of modern society and play a crucial role in the development of the traditional Chinese medicine preparation industry. Description of the drawings

[0035] Figure 1 It is the experimental result of the compound preparation for improving anemia;

[0036] Figure 2 It is the experimental result of the compound preparation for improving microcirculation function. Detailed implementation manners

[0037] The following is described in conjunction with specific embodiments:

[0038] Description of the source of experimental materials:

[0039] Phenylhydrazine, arachidonic acid, vinorelbine: Purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

[0040] AB-strain zebrafish, transgenic zebrafish (Tg: zlyz-EGFP): Provided by the Zebrafish Drug Screening Platform of the Shandong Academy of Sciences.

[0041] Example 1: Preparation of a compound preparation

[0042] (1) Prepare the basic formula according to the weight ratio: 15 parts of ginseng, 20 parts of rehmannia root, 30 parts of codonopsis pilosula, 20 parts of atractylodes macrocephala, 15 parts of hawthorn; dry and pulverize the medicinal materials and mix them evenly to obtain medicinal material powder; mix phenylalanine, glucose and water in a molar ratio of 1:2:3, heat and stir at 85 °C for 1 h to prepare a binary eutectic solvent; add the binary eutectic solvent to 1 kg of medicinal material powder at a ratio of 1:15 (g / mL), perform ultrasonic extraction at 70 °C for 2 h, filter to obtain extract 1 and medicinal material filter residue.

[0043] (2) Add AB-8 macroporous resin to extract 1 in step (1) at a ratio of 1:6 (g / mL), shake at 100 r / min on a shaker at room temperature for 10 h until fully adsorbed, take out the macroporous resin to obtain an adsorption solution; desorb the taken-out macroporous resin with 10 times the volume of 95% (v / v) ethanol to obtain a desorption solution, concentrate and dry the desorption solution to obtain extract 1.

[0044] (3) Add lactic acid to the adsorption solution in step (2), where the molar ratio between phenylalanine and lactic acid is 1:1, heat and stir at 85 °C for 1 h to form a ternary eutectic solvent from the binary eutectic solvent in the adsorption solution and hydrogen bond donor B, use the ternary eutectic solvent to perform ultrasonic extraction on the medicinal material filter residue in step (1) at 70 °C for 2 h, filter to obtain extract 2; add 5 times the volume of absolute ethanol to extract 2, perform alcohol precipitation for 12 h, centrifuge, and dry the precipitate after centrifugation to obtain extract 2.

[0045] (4) Take 3 parts by weight of donkey-hide gelatin and ultrafinely pulverize it to 1000 meshes, mix it with extracts 1 and 2 to obtain 456 g of the compound preparation.

[0046] Example 2: Preparation of a compound preparation

[0047] (1) Prepare the basic formula according to the proportion by weight: 25 parts of ginseng, 30 parts of rehmannia root, 25 parts of codonopsis pilosula, 10 parts of atractylodes macrocephala, and 10 parts of hawthorn; dry and pulverize the medicinal materials and mix them evenly to obtain medicinal material powder; mix aspartic acid, ethylene glycol, and water in a molar ratio of 1:3:4, heat and stir at 90 °C for 0.5 h to prepare a binary eutectic solvent; add the binary eutectic solvent to 1 kg of medicinal material powder at a ratio of 1:25 (g / mL), perform ultrasonic extraction at 80 °C for 0.5 h, and filter to obtain extraction solution 1 and medicinal material residue.

[0048] (2) Add AB-8 macroporous resin to extraction solution 1 in step (1) at a ratio of 1:8 (g / mL), shake on a shaker at 60 r / min at room temperature for 12 h until fully adsorbed, take out the macroporous resin to obtain an adsorption solution; desorb the taken-out macroporous resin with 8 times the volume of 95% (v / v) ethanol to obtain a desorption solution, concentrate and dry the desorption solution to obtain extract 1.

[0049] (3) Add citric acid to the adsorption solution in step (2), where the molar ratio between aspartic acid and citric acid is 1:2, heat and stir at 90 °C for 0.5 h to form a ternary eutectic solvent from the binary eutectic solvent and hydrogen bond donor B in the adsorption solution, use the ternary eutectic solvent to perform ultrasonic extraction on the medicinal material residue in step (1) at 80 °C for 0.5 h, and filter to obtain extraction solution 2; add 6 times the volume of absolute ethanol to extraction solution 2, perform alcohol precipitation for 10 h, centrifuge, and dry the precipitate after centrifugation to obtain extract 2.

[0050] (4) Take 5 parts by weight of donkey-hide gelatin, ultrafinely pulverize it to 800 meshes, and mix it evenly with extracts 1 and 2 to obtain 459 g of a compound preparation.

[0051] Example 3: Preparation of a compound preparation

[0052] (1) Prepare the basic formula according to the proportion by weight: 10 parts of ginseng, 10 parts of rehmannia root, 30 parts of codonopsis pilosula, 30 parts of atractylodes macrocephala, and 20 parts of hawthorn; dry and pulverize the medicinal materials and mix them evenly to obtain medicinal material powder; mix betaine, 1,4-butanediol, and water in a molar ratio of 1:1:2, heat and stir at 75 °C for 2 h to prepare a binary eutectic solvent; add the binary eutectic solvent to 1 kg of medicinal material powder at a ratio of 1:10 (g / mL), perform ultrasonic extraction at 60 °C for 3 h, and filter to obtain extraction solution 1 and medicinal material residue.

[0053] (2) Add AB-8 macroporous resin to extraction solution 1 in step (1) at a ratio of 1:5 (g / mL), shake on a shaker at 120 r / min at room temperature for 8 h until fully adsorbed, take out the macroporous resin to obtain an adsorption solution; desorb the taken-out macroporous resin with 12 times the volume of 95% (v / v) ethanol to obtain a desorption solution, concentrate and dry the desorption solution to obtain extract 1.

[0054] (3) Add malonic acid to the adsorption solution in step (2), where the molar ratio between betaine and malonic acid is 1:3. Heat and stir at 75 °C for 2 h to form a ternary deep eutectic solvent from the binary deep eutectic solvent in the adsorption solution and the hydrogen bond donor B. Use the ternary deep eutectic solvent to ultrasonically extract the medicinal residues in step (1) at 60 °C for 3 h, filter to obtain extract 2; add 4 times the volume of absolute ethanol to extract 2, perform alcohol precipitation for 14 h, centrifuge, and dry the precipitate after centrifugation to obtain extract 2.

[0055] (4) Take 1 part by weight of donkey-hide gelatin and ultrafinely pulverize it to 1200 mesh, mix it with extracts 1 and 2 to obtain 435 g of the compound preparation.

[0056] Comparative Example 1: Preparation of a compound preparation

[0057] Different from Examples 1 to 3, this comparative example uses a traditional water decoction extraction method, and the specific operation steps are as follows:

[0058] (1) Prepare the basic formula according to the weight ratio: 15 parts of ginseng, 20 parts of rehmannia root, 30 parts of codonopsis pilosula, 20 parts of atractylodes macrocephala, 15 parts of hawthorn; dry and pulverize the medicinal materials and mix them evenly to obtain medicinal material powder; add water to 1 kg of the medicinal material powder according to a ratio of 1:30 (g / mL), decoct and extract for 1 h to obtain an extract, concentrate and dry the extract to obtain an extract.

[0059] (2) Take 3 parts by weight of donkey-hide gelatin and ultrafinely pulverize it to 1000 mesh, mix it with the extract in step (1) to obtain 334 g of the compound preparation.

[0060] Comparative Example 2: Preparation of a compound preparation

[0061] Different from Examples 1 to 3, a water / organic solvent extraction method is used, and the specific operation steps are as follows:

[0062] (1) Prepare the basic formula according to the weight ratio: 15 parts of ginseng, 20 parts of rehmannia root, 30 parts of codonopsis pilosula, 20 parts of atractylodes macrocephala, 15 parts of hawthorn; dry and pulverize the medicinal materials and mix them evenly to obtain medicinal material powder; add water to 1 kg of the medicinal material powder according to a ratio of 1:30 (g / mL), decoct and extract for 1 h to obtain extract A, filter extract A to obtain a filtrate and residues, concentrate and dry the filtrate to obtain extract A.

[0063] (2) Add absolute ethanol to the residues in step (1) according to a ratio of 1:20 (g / mL), extract at 80 °C for 1 h to obtain extract B, filter extract B to collect the filtrate, and concentrate and dry the filtrate to obtain extract B.

[0064] (3) Take 3 parts by weight of donkey-hide gelatin and ultrafinely pulverize it to 1000 meshes, mix it with extract A in step (1) and extract B in step (2) to obtain 380 g of the compound preparation.

[0065] Comparative Example 3: Preparation of a compound preparation

[0066] Different from Example 1, binary eutectic solvents composed of phenylalanine and glucose were used for both extractions, that is: in step (3), lactic acid was not added, and the binary eutectic solvent in the adsorption solution was continued to be used to ultrasonically extract the medicinal residues in step (1) at 70 °C for 2 h; the remaining steps were the same as those in Example 1 to obtain 383 g of the compound preparation.

[0067] Experimental Example 1: Detection of the contents of polysaccharides, saponins, and flavonoids in the compound preparation

[0068] (1) Drawing of the polysaccharide standard curve: Respectively measure 1.0 mL, 1.5 mL, 2.0 mL, 2.5 mL, and 3.0 mL of glucose reference substance solutions with a concentration of 0.6 mg / mL, place them in 50 mL volumetric flasks respectively, add water to a constant volume of 50 mL, and mix well. Pipette 2 mL of each concentration of glucose solution, add 1 mL of 5% (w / v) phenol solution, and then quickly add 7 mL of sulfuric acid. After taking it out in a boiling water bath for 15 min, place it in a cold water bath for 5 min to terminate the reaction to obtain the reaction solution. Measure the absorbance value of the reaction solution at a wavelength of 490 nm and draw the standard curve to obtain the linear equation: Y = 5.8653X + 0.0406 (r = 0.9982).

[0069] (2) Drawing of the saponin standard curve: Respectively measure 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, and 0.5 mL of oleanolic acid reference substance solutions with a concentration of 0.3 mg / mL. After evaporating the solvent to dryness, add 10 mL of perchloric acid and react in a constant temperature water bath at 70 °C for 15 min to obtain the reaction solution. Measure the absorbance value of the reaction solution at a wavelength of 408 nm and draw the standard curve to obtain the linear equation: Y = 2.5413X + 0.0326 (r = 0.9967).

[0070] (3) Drawing of the flavonoid standard curve: Respectively measure 0.1 mL, 0.2 mL, 0.4 mL, 0.6 mL, and 0.8 mL of quercetin reference substance solutions with a concentration of 0.1 mg / mL, place them in 25 mL volumetric flasks respectively. Add 1 mL of NaNO2 solution with a concentration of 0.50 mol / L and 1 mL of AlCl3 solution with a concentration of 0.30 mol / L to each concentration of quercetin solution, shake well and then add 6 mL of 1 mol / L NaOH solution, and make up to 25 mL with 70% (v / v) ethanol to obtain the reaction solution. Measure the absorbance value of the reaction solution at a wavelength of 525 nm and draw the standard curve to obtain the linear equation: Y = 4.8715X - 0.0026 (r = 0.9991).

[0071] (4) Content determination: Weigh 5 mg of the compound preparation, and ultrasonically extract it with 25 mL of water, 30% ethanol, and 70% ethanol for 30 min respectively to obtain a polysaccharide sample solution, a saponin sample solution, and a flavonoid sample solution. Pipette 1 mL of each sample solution and place them in graduated colorimetric test tubes, measure the absorbance values at specific wavelengths, and then calculate the contents (%) of polysaccharides, saponins, and flavonoids in the compound preparation according to the standard curve.

[0072] The measurement results are shown in Table 1:

[0073] Table 1. Content determination results

[0074]

[0075]

[0076] It can be seen from Table 1 that the yields of the compound preparations obtained in Examples 1 - 3 can reach over 43%, and the contents of the effective active ingredients polysaccharides, saponins, and flavonoids can reach 26%, 23%, and over 9% respectively; in Comparative Example 1, the traditional water decoction extraction method was used, and the results showed that the content of flavonoid components decreased significantly compared with Examples 1 - 3; in Comparative Example 2, the water / alcohol solvent sequential extraction technology was used, and its extraction effects in all aspects were also weaker than those in Examples 1 - 3; in Comparative Example 3, only the binary eutectic solvent was used to prepare the compound components, and it was detected that the content of polysaccharide components was relatively low. The above experimental results show that compared with the existing traditional technologies, the ternary eutectic solvent extraction technology provided by the present invention has the dual advantages of large acquisition amount and high enrichment rate of pharmacodynamic components in the process of preparing the compound preparation for supplementing qi and generating blood with ginseng, rehmannia root, codonopsis pilosula, atractylodes macrocephala, and hawthorn.

[0077] Experimental Example 2: Experiment on the compound preparation for improving anemia

[0078] Experimental grouping: blank group, model group (0.1 μg / mL phenylhydrazine), low-dose administration group of Example 1 (0.1 μg / mL phenylhydrazine + 2.5 μg / mL compound preparation of Example 1), medium-dose administration group of Example 1 (0.1 μg / mL phenylhydrazine + 5 μg / mL compound preparation of Example 1), high-dose administration group of Example 1 (0.1 μg / mL phenylhydrazine + 10 μg / mL compound preparation of Example 1), administration group of Comparative Example 1 (0.1 μg / mL phenylhydrazine + 10 μg / mL compound preparation of Comparative Example 1), and administration group of Comparative Example 2 (0.1 μg / mL phenylhydrazine + 10 μg / mL compound preparation of Comparative Example 2).

[0079] Experimental method: Healthy wild-type AB zebrafish at 56 hpf of development were selected. After the embryos hatched, they were transferred to a 24-well plate, with 10 fish in each well. The zebrafish were treated according to the above experimental groups respectively, and then cultured in a constant temperature incubator at 28.5 °C until 3 dpf. The o-dianisidine staining method was used to stain the red blood cells of zebrafish in each group, and the staining duration was 15 min. After staining, observation and photography were carried out under a microscope. The integrated optical density (IOD) of red blood cell staining of zebrafish in each group was measured with ImageJ software, and relevant charts were made and statistical analysis was carried out.

[0080] The experimental results are as Figure 1 shown; it can be Figure 1 seen that the low, medium, and high-dose administration groups of Example 1 can all alleviate the anemia condition in the zebrafish in vivo model caused by phenylhydrazine, and the data show a dose-effect relationship; especially when the administration concentration is 10 μg / mL, the compound preparation has the best improvement effect on the integrated optical density of red blood cells (close to 2×10 7 ), proving that the compound preparation prepared in Example 1 can exert excellent blood-tonifying activity.

[0081] At a concentration of 10 μg / mL, the compound preparation prepared by the traditional water decoction method in Comparative Example 1 has certain anti-anemic activity, but its effect is significantly weaker than that of Example 1; the compound preparation prepared by the water / organic solvent extraction method in Comparative Example 2 has an improved anti-anemic activity compared with the traditional water decoction method, but it is also weaker than the compound preparation prepared in Example 1 at the same concentration. The above experimental results show that the extraction method provided by the present invention can exert the maximum therapeutic efficacy of the traditional Chinese medicine formula compared with the traditional extraction technology. There have been a large number of reports on the application of components such as polysaccharides, saponins, and flavonoids in modern medicine for enriching blood and promoting blood circulation. Therefore, the reason why the compound preparation of the present invention has a good therapeutic effect on anemia symptoms may be related to the relatively complete extraction of active ingredients.

[0082] Experimental Example 3: Experiment on the promotion of microcirculation function by the compound preparation

[0083] Experimental grouping: blank group, model group, aspirin group (20 μg / mL aspirin), low-dose administration group of Example 1 (5 μg / mL compound preparation of Example 1), medium-dose administration group of Example 1 (10 μg / mL compound preparation of Example 1), high-dose administration group of Example 1 (20 μg / mL compound preparation of Example 1), administration group of Comparative Example 1 (20 μg / mL compound preparation of Comparative Example 1), and administration group of Comparative Example 2 (20 μg / mL compound preparation of Comparative Example 2).

[0084] Experimental method: When the fertilized eggs developed to 3 dpf, normal zebrafish embryos were selected under a stereomicroscope and transferred into a 24-well plate, with 10 embryos in each well. Except for the blank group and the model group, the zebrafish embryos were treated with drugs according to the above experimental groups respectively. After being placed in an incubator at 28 °C for 6 h, except for the blank group, arachidonic acid with a final concentration of 80 μM was added to each group for modeling, and then all were taken out after being incubated in an incubator at 28 °C for 1 day. The zebrafish were fixed on a glass slide laterally (with two eyes overlapping) with methylcellulose, and their arterial blood flow velocity was recorded under a fluorescence microscope; zebrblood TM (ViewPoint, Lyon, France) was used to analyze the blood flow video, and the effects of zebrafish arterial blood flow velocity in each group were statistically analyzed, and the hemodynamic data were quantitatively evaluated.

[0085] The experimental results are as Figure 2 shown; it can be Figure 2 seen that the low, medium, and high-dose administration groups of Example 1 can all repair the slowdown of zebrafish arterial blood flow caused by arachidonic acid. Research reports show that increasing the blood flow of arterioles can effectively enhance the blood perfusion of body tissues, thus playing a role in improving microcirculation. In addition, the compound preparation of Example 1 can also well increase the diameter of zebrafish arterial blood vessels and shows a dose-dependence, which is another functional way to improve microcirculation. Compared with the traditional western medicine aspirin, at the same concentration of 20 μg / mL, the effect of the compound preparation of Example 1 on enhancing arterial blood flow velocity is basically the same as that of the aspirin group, and its dilating effect on blood vessel diameter is even stronger than that of the aspirin administration group, which also reflects the advantages and characteristics of the microcirculation-improving effect of this compound preparation in clinical applications.

[0086] In Comparative Example 1 and Comparative Example 2, the compound preparations were prepared by water decoction and water / organic solvent extraction methods respectively, and the two indicators of arterial blood flow velocity and arterial blood vessel diameter were weaker than those of the Example group and the aspirin group. The above experimental results show that compared with the existing traditional technologies, the ternary eutectic solvent extraction technology provided by the present invention can obtain a compound preparation with more efficient microcirculation-improving ability during the extraction process of the qi-invigorating and blood-generating formula of ginseng, rehmannia root, codonopsis pilosula, atractylodes macrocephala, and hawthorn.

[0087] Experimental Example 4: Immunomodulatory experiment of the compound preparation

[0088] Macrophages have active phagocytic functions and can remove antigenic substances and degenerated cells in the body, playing an important role in both specific and non-specific immunity.

[0089] (1) Immune enhancement experiment

[0090] The chemotherapy drug vinorelbine has obvious myelosuppression, which can significantly reduce the number of platelets, red blood cells and macrophages in the body, and ultimately lead to a decrease in immunity. Therefore, in this experiment, a zebrafish model with low immunity was constructed using vinorelbine.

[0091] Experimental grouping: blank control group, model group (150 μg / mL vinorelbine), low-dose administration group of Example 1 (150 μg / mL vinorelbine + 2.5 μg / mL compound preparation of Example 1), medium-dose administration group of Example 1 (150 μg / mL vinorelbine + 5 μg / mL compound preparation of Example 1), high-dose administration group of Example 1 (150 μg / mL vinorelbine + 10 μg / mL compound preparation of Example 1), administration group of Comparative Example 1 (150 μg / mL vinorelbine + 10 μg / mL compound preparation of Comparative Example 1), and administration group of Comparative Example 2 (150 μg / mL vinorelbine + 10 μg / mL compound preparation of Comparative Example 2).

[0092] Experimental method: When the fertilized eggs developed to 2 dpf, normal transgenic zebrafish (Tg: zlyz-EGFP) embryos were selected under a stereomicroscope for dechorionation treatment, and then transferred into a 24-well plate, with 10 embryos in each well; the zebrafish were treated according to the above experimental groupings respectively, and the culture water was added to 2.0 mL; then the 24-well plate was placed in an incubator at 28 °C and incubated for another 24 h, and the macrophages in the tails of the zebrafish were photographed and counted using a fluorescence microscope.

[0093] (2) Anti-inflammatory activity experiment

[0094] CuSO4 can induce the death of hair cells in the lateral line neuromas of zebrafish, and this damage can lead to a strong local inflammatory response in the neuromas, including the infiltration of macrophages and neutrophils.

[0095] Experimental grouping: blank control group, model group, low-dose administration group of Example 1 (2.5 μg / mL compound preparation of Example 1), medium-dose administration group of Example 1 (5 μg / mL compound preparation of Example 1), high-dose administration group of Example 1 (10 μg / mL compound preparation of Example 1), administration group of Comparative Example 1 (10 μg / mL compound preparation of Comparative Example 1), and administration group of Comparative Example 2 (10 μg / mL compound preparation of Comparative Example 2).

[0096] Experimental method: When the fertilized eggs developed to 2 dpf, normal transgenic zebrafish (Tg: zlyz-EGFP) embryos were selected under a stereomicroscope for dechorionation treatment, and then transferred into 24-well plates, with 10 embryos in each well. Except for the blank group and the model group, the zebrafish embryos were treated with drugs according to the above experimental groups respectively, and the culture water was added to 2.0 mL. Then, the 24-well plates were placed in an incubator at 28 °C and incubated for another 2 h. Except for the blank group, CuSO4 with a final concentration of 20 μM was added to each group for modeling, and then all were placed in an incubator at 28 °C for light-avoiding culture for 1 h. The macrophage inflammatory response was observed under a fluorescence microscope, and the number of macrophages around the lateral line neuromasts was counted.

[0097] (3) The experimental results are shown in Table 2, where *p < 0.05, **p < 0.01 (compared with the model group).

[0098] Table 2. Effects of the compound preparation on immunomodulation of the zebrafish drug screening model

[0099]

[0100] It can be seen from Table 2 that:

[0101] In the immune enhancement experiment, compared with the model group, the low, medium, and high-dose administration groups of Example 1 had a good effect on increasing the number of macrophages in the tails of zebrafish, and the medium and high-dose administration groups showed statistical differences. At the same test concentration (10 μg / mL), the number of macrophages in Comparative Example 1 and Comparative Example 2 increased to a certain extent compared with the model group, but the recovery effect was significantly weaker than that of Example 1. The above results indicate that the compound preparation provided by the present invention has a good immune enhancement effect.

[0102] In the anti-inflammatory activity experiment, compared with the model group, the low, medium, and high-dose administration groups of Example 1 showed a good inhibitory effect on macrophage migration in zebrafish, and the medium and high-dose administration groups showed statistical differences. The compound preparation prepared in Comparative Example 1 showed no statistical difference compared with the model group at a concentration of 10 μg / mL, and the anti-inflammatory effect of the compound preparation prepared in Comparative Example 2 was also significantly weaker than that of Example 1. The above results indicate that the compound preparation provided by the present invention can not only enhance the immune function of macrophages but also regulate the migration and aggregation of macrophages at the inflammatory site, has excellent bidirectional regulation function, and effectively reflects the traditional Chinese medicine treatment concept of multi-target, multi-link, and overall regulation to restore balance.

[0103] Experimental Example 5: Tyrosinase inhibition experiment of the compound preparation

[0104] Melanin is formed from tyrosine under the action of tyrosinase. Therefore, specifically inhibiting the activity of tyrosinase and reducing its ability to catalyze the conversion of tyrosine into melanin can reduce the production of melanin, thereby achieving the effect of skin whitening.

[0105] Preparation of L-tyrosine solution: Take 0.05 g of L-tyrosine, dissolve it in 35 mL of 0.1 mol / L hydrochloric acid, and then add PBS buffer solution with pH = 6.8 to make up the volume to 100 mL.

[0106] Preparation of tyrosinase solution: Dissolve 25000 U of tyrosinase in 250 mL of pure water to obtain a tyrosinase solution with a concentration of 100 U / mL.

[0107] Using the compound preparations with different concentrations prepared in Example 1 and Comparative Examples 1 - 3 as tyrosinase inhibitors, add L-tyrosine solution, compound preparation (test sample), and PBS buffer solution into a test tube according to the dosage in Table 3 below. After mixing, preheat the test tube in a 37°C water bath for 15 min, and then add the tyrosinase solution; take it out after reacting at a constant temperature of 37°C for 10 min, and measure the absorbance value of the reaction solution at a wavelength of 475 nm with a spectrophotometer.

[0108] Table 3. Tyrosinase Activity Inhibition Test

[0109] Reagent A1 A2 B1 B2 L-Tyrosine solution (mL) 2 2 2 2 Test sample (mL) 0 0 2 2 PBS buffer solution (mL) 4 5 2 3 Tyrosinase solution (mL) 1 0 1 0

[0110] Calculate the tyrosinase inhibition rate according to the following formula:

[0111] Among them, A1, A2, B1, and B2 respectively represent the absorbance values of the reaction solutions of each group at a wavelength of 475 nm.

[0112] The experimental results are shown in Table 4 below:

[0113] Table 4. Tyrosinase Activity Inhibition Rate of Compound Preparation

[0114]

[0115] It can be seen from Table 4 that the compound preparation prepared in Example 1 has the most excellent tyrosinase inhibition activity, while the compound preparation prepared by the traditional water decoction extraction method in Comparative Example 1 has the weakest tyrosinase inhibition activity. It has been reported that plant flavonoids are a class of natural tyrosinase inhibitory components with excellent activity. Therefore, the reason for the weak activity of Comparative Example 1 may be related to the low extraction rate of flavonoid components therein. In addition, the tyrosinase inhibition activity of Example 1 is also significantly stronger than that of Comparative Example 2 and Comparative Example 3. From the experimental results, it is speculated that in addition to the influencing factor of component content, the tyrosinase inhibition activity may also be related to the multi-target synergistic effect of each component in the compound preparation prepared in Example 1.

Claims

1. A preparation method of a compound preparation, characterized in that, Specifically, it includes the following steps: (1) Prepare the basic formula according to the weight parts ratio: 5 - 25 parts of ginseng, 10 - 30 parts of rehmannia root, 20 - 35 parts of codonopsis pilosula, 10 - 30 parts of atractylodes macrocephala, 5 - 25 parts of hawthorn; dry and crush the medicinal materials and mix them evenly to obtain medicinal material powder. Add a binary eutectic solvent composed of a hydrogen bond acceptor, a hydrogen bond donor A, and water to the medicinal material powder, and perform ultrasonic extraction at 60 - 80 °C for 0.5 - 3 h, then filter to obtain extraction solution 1 and medicinal material residue; (2) Add AB-8 macroporous resin to the extraction solution 1 in step (1), oscillate at 50 - 130 r / min at room temperature for 8 - 12 h. After taking out the macroporous resin, obtain the adsorption solution; desorb the taken-out macroporous resin with 95% (v / v) ethanol to obtain the desorption solution, concentrate and dry the desorption solution to obtain extract 1; (3) Add hydrogen bond donor B to the adsorption solution in step (2) so that the binary eutectic solvent in the adsorption solution forms a ternary eutectic solvent with hydrogen bond donor B. Use the ternary eutectic solvent to perform ultrasonic extraction on the medicinal material residue in step (1) at 60 - 80 °C for 0.5 - 3 h, then filter to obtain extraction solution 2; add 4 - 6 times the volume of absolute ethanol to extraction solution 2, perform alcohol precipitation for 10 - 14 h, centrifuge, and dry the precipitate after centrifugation to obtain extract 2; (4) Take 1 - 5 weight parts of donkey-hide gelatin, crush it, and mix it evenly with extracts 1 and 2 to obtain a compound preparation; Among them, the hydrogen bond acceptor is one of betaine, aspartic acid, and phenylalanine, the hydrogen bond donor A is one of ethylene glycol, 1,4-butanediol, and glucose, and the hydrogen bond donor B is one of citric acid, lactic acid, and malonic acid; The compound preparation is used for preparing drugs for treating anemia, improving microcirculation, enhancing immunity, anti-inflammatory drugs, and whitening drugs.

2. The preparation method according to claim 1, characterized in that, In the binary eutectic solvent in step (1), the hydrogen bond acceptor is one of betaine, aspartic acid, and phenylalanine, and the hydrogen bond donor A is one of ethylene glycol, 1,4-butanediol, and glucose; the molar ratio among the hydrogen bond acceptor, hydrogen bond donor A, and water is 1: (0.5 - 3.5): (1.5 - 5); the binary eutectic solvent is prepared by mixing the hydrogen bond acceptor, hydrogen bond donor A, and water, and heating and stirring at 75 - 90 °C for 0.5 - 3 h.

3. The preparation method according to claim 1, wherein In the binary eutectic solvent in step (1), the hydrogen bond acceptor is one of aspartic acid and phenylalanine, and the hydrogen bond donor A is one of ethylene glycol and glucose; the molar ratio among the hydrogen bond acceptor, hydrogen bond donor A, and water is 1: (1 - 3): (2 - 4.5); the binary eutectic solvent is prepared by mixing the hydrogen bond acceptor, hydrogen bond donor A, and water, and heating and stirring at 80 - 90 °C for 1 - 2 h.

4. The preparation method according to claim 1, characterized in that, In the binary eutectic solvent in step (1), the hydrogen bond acceptor is phenylalanine, and the hydrogen bond donor A is glucose; the molar ratio among the hydrogen bond acceptor, hydrogen bond donor A, and water is 1:2:3; the binary eutectic solvent is prepared by mixing the hydrogen bond acceptor, hydrogen bond donor A, and water, and heating and stirring at 85 °C for 1 h.

5. The preparation method according to claim 1, characterized in that, The ratio between the medicinal material powder and the binary eutectic solvent in step (1) is 1: (5 - 25) (g / mL).

6. The preparation method according to claim 1, characterized in that, In step (2), the ratio of the AB-8 macroporous resin to the extraction solution 1 is 1:(3 - 10) (g / mL), and the amount of 95% (v / v) ethanol used is 5 - 15 times the volume of the AB-8 macroporous resin.

7. The preparation method according to claim 1, wherein, In step (2), the ratio of the AB-8 macroporous resin to the extraction solution 1 is 1:(5 - 8) (g / mL), and the amount of 95% (v / v) ethanol used is 8 - 12 times the volume of the AB-8 macroporous resin.

8. The preparation method according to claim 1, characterized in that, In step (2), the ratio of the AB-8 macroporous resin to the extraction solution 1 is 1:6 (g / mL), and the amount of 95% (v / v) ethanol used is 10 times the volume of the AB-8 macroporous resin.

9. The preparation method according to claim 1, wherein In the ternary eutectic solvent described in step (3), the hydrogen bond donor B is one of citric acid, lactic acid, and malonic acid; the molar ratio between the hydrogen bond acceptor and the hydrogen bond donor B is 1:(1 - 2); the ternary eutectic solvent is prepared by adding the hydrogen bond donor B to the adsorption solution and heating and stirring at 80 - 90 °C for 1 - 2 h.

10. The preparation method according to claim 1, characterized in that, In the ternary eutectic solvent described in step (3), the hydrogen bond donor B is lactic acid; the molar ratio between the hydrogen bond acceptor and the hydrogen bond donor B is 1:1; the ternary eutectic solvent is prepared by adding the hydrogen bond donor B to the adsorption solution and heating and stirring at 85 °C for 1 h.

11. The preparation method according to claim 1, characterized in that, In step (4), the pulverization particle size of the donkey-hide gelatin is 500 - 1500 mesh.

12. The preparation method according to claim 1, wherein, In step (4), the pulverization particle size of the donkey-hide gelatin is 800 - 1200 mesh.

13. The preparation method according to claim 1, characterized in that, In step (4), the pulverization particle size of the donkey-hide gelatin is 1000 mesh.

14. A compound preparation prepared by the preparation method according to any one of claims 1 - 13.

15. Use of the compound preparation according to claim 14 in the preparation of drugs for treating anemia, improving microcirculation, enhancing immunity, anti-inflammatory drugs, and whitening drugs.

Images