Purification method of total phenolic acid from blueberry and its application in medicine

Abstract

The invention provides a method for purifying total phenolic acid of blue cloth and its application in medicine, relates to the technical field of traditional Chinese medicine, and specifically relates to the application of total phenolic acid of blue cloth in preparing medicine for promoting wound healing and inhibiting scar formation; total phenolic acid of blue cloth is obtained by a purification method comprising the following steps: (1) a sample solution containing a blue cloth extract is loaded on a macroporous adsorption resin column with a resin bed diameter height ratio of 1:4-12 at a flow rate of 2-4BV / h; (2) a 5-20% ethanol solution is used for impurity removal and elution, the flow rate of impurity removal and elution is 2-4BV / h, and the amount of dilute ethanol solution is 2-6BV; (3) a 50-80% ethanol solution is used for elution, and the elution flow rate is 2-6BV / h to obtain total phenolic acid of blue cloth. The medicine prepared in the application has the effect of significantly improving the relative wound healing rate and the relative scar inhibition rate.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a method for purifying total phenolic acid of bluebutan and application thereof in medicines. Background Art

[0002] Lanbuzheng has the effects of invigorating qi and strengthening spleen, nourishing blood and yin, moistening lungs and resolving phlegm, and is used to treat qi and blood deficiency, consumptive cough, spleen deficiency and leukorrhea, etc. As a medicinal material, Lanbuzheng contains a variety of chemical components such as triterpenes, flavonoids, tannins, lignans, and volatile oils. Among them, phenolic acid compounds are mainly tannin total phenolic acids, which have strong antioxidant and angiogenesis activities.

[0003] Chinese invention patent CN108969580A discloses a preparation method and application of total tannins from blue cloth. The preparation method is as follows: (1) taking blue cloth medicinal material, crushing it into powder and then soaking it in acetone; (2) performing ultrasonic extraction; (3) extracting it with ethyl acetate saturated with 2 times the volume of water; (4) adding gelatin solution for precipitation; (5) performing ultrasonic extraction with acetone again; (6) vacuum drying to obtain purified blue cloth tannins. In the invention, the preparation method of total tannins from blue cloth includes the steps of ultrasonic extraction, solvent extraction, gelatin precipitation, vacuum drying, etc. The prepared purified blue cloth tannins are effective in replenishing qi and blood. However, the preparation method uses a large amount of organic solvents, such as acetone and ethyl acetate, which have certain toxicity. And the content and transfer rate of the purified total tannins are low.

[0004] At present, there are few studies on the preparation and purification technology of tannin phenolic acid from blue cloth. Most of the commonly used methods for extracting and purifying tannin phenolic acid from other medicinal materials use ethanol extraction, followed by adsorption, elution and purification with the help of macroporous adsorption resin. Chinese invention patent CN108338996A discloses a method for preparing tannin active ingredients in Terminalia chebula, comprising the following steps: ultrasonic extraction of Terminalia chebula powder with anhydrous ethanol, and decompression and concentration of the extract to form an extract; dissolving the extract with an appropriate amount of water and then separating it with macroporous adsorption resin column chromatography, eluting with pure water, ethanol with a volume fraction of 20%, 40%, 60%, 80%, 90%, and 95% in sequence, collecting the 90% ethanol elution portion, and then separating it with silica gel column chromatography, eluting with chloroform-methanol with a volume ratio of 65:1, 55:1, 45:1, 35:1, 25:1, and 10:1 in sequence, collecting the 25:1 elution portion, and concentrating it. The macroporous adsorption resin column used in this technology is D101 type, and toxic chloroform-methanol is also used for elution, which limits the application of tannin active ingredients in drug preparation.

[0005] Chinese invention patent CN113332320A discloses a purification method of terminalia chebula tannin part and terminalia chebula tannin part, the purification method comprises the following steps: loading a sample solution containing terminalia chebula extract on a chromatography column equipped with HPD-400 macroporous resin for adsorption; after adsorption, eluting with deionized water at a flow rate of 2.5-4.5BV / h; then eluting with 60-80vol% ethanol at a flow rate of 5-7BV / h, collecting the ethanol eluate, concentrating it, and drying it to obtain a terminalia chebula tannin part purification product. The terminalia chebula tannin part obtained by the invention has a total tannin content of more than 60%, and a total tannin transfer rate of more than 60%. The total tannin transfer rate of this technology is relatively low.

[0006] Based on the above technical problems, further research is needed on the extraction and purification of total phenolic acids from blue cloth tannins to improve the adsorption rate, content and transfer rate of total phenolic acids from blue cloth. At the same time, further research is needed on the anti-inflammatory, antibacterial, antioxidant and other activities of total phenolic acids from blue cloth tannins to broaden their huge application potential in pharmaceutical applications. Summary of the invention

[0007] The present invention aims at the problems existing in the prior art and provides a method for purifying total phenolic acid of blue cloth and its application in medicine. The blue cloth extract after ethanol extraction is purified by using a macroporous adsorption resin, the sample solution concentration is controlled to be 4-8 mg / mL, and the sample solution is passed into a macroporous adsorption resin column with a resin bed diameter height ratio of 1:4-12 at a flow rate of 2-4 BV / h, the sample amount is ≤3 BV, and the impurities are eluted and removed with a dilute ethanol solution, and the impurity removal flow rate is controlled at 2-4 BV / h. After the impurities are removed, the elution is carried out with a high concentration of ethanol solution, and the elution flow rate is controlled at 2-6 BV / h, and finally purified blue cloth total phenolic acid is obtained; the obtained blue cloth total phenolic acid is used to prepare a drug that promotes wound healing and inhibits scar formation, and the prepared drug has the effect of significantly improving the relative wound healing rate and the relative scar inhibition rate.

[0008] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0009] First, the present invention provides an application of total phenolic acid of blue cloth in preparing a drug for promoting wound healing and inhibiting scar formation; the total phenolic acid of blue cloth is obtained by a purification method comprising the following steps:

[0010] (1) Loading the sample solution containing the blue cloth extract onto a macroporous adsorption resin column with a resin bed diameter-to-height ratio of 1:4-12 at a flow rate of 2-4 BV / h for adsorption;

[0011] (2) Impurity removal: Use a dilute ethanol solution with a volume concentration of 5-20% for impurity removal elution. The flow rate of the impurity removal elution is 2-4BV / h, and the amount of the dilute ethanol solution for impurity removal elution is controlled to be 2-6BV;

[0012] (3) Elution: Use a concentrated ethanol solution with a volume concentration of 50-80% for elution, control the elution flow rate to 2-6BV / h, collect the ethanol eluate, and obtain the total phenolic acid of blue cloth.

[0013] Preferably, in step (1), the loading solution is an aqueous solution of indigofera lancifolia extract, and the concentration is 4-10 mg / mL; the concentration includes any real number and endpoint value within the numerical range, for example 4 mg / mL, 4.5 mg / mL, 5 mg / mL, 5.5 mg / mL, 6 mg / mL, 6.5 mg / mL, 7 mg / mL, 7.5 mg / mL, 8 mg / mL, 8.5 mg / mL, 9 mg / mL, 9.5 mg / mL, 10 mg / mL.

[0014] Further preferably, the concentration of the loading solution is 8 mg / mL.

[0015] Preferably, in step (1), the flow rate is 2-4 BV / h, which includes any real number and endpoint value within the numerical range, for example 2 BV / h, 2.2 BV / h, 2.5 BV / h, 2.8 BV / h, 3 BV / h, 3.2 BV / h, 3.5 BV / h, 3.8 BV / h, 4 BV / h.

[0016] Further preferably, the flow rate is 2BV / h.

[0017] Preferably, in step (1), the macroporous adsorption resin is selected from one of HPD-400 macroporous adsorption resin, HPD-600 macroporous adsorption resin, AB-8 macroporous adsorption resin and D101 macroporous adsorption resin.

[0018] Further preferably, the macroporous adsorption resin is HPD-400 macroporous adsorption resin.

[0019] Preferably, in step (1), the resin bed diameter height ratio is 1:4-12; the ratio includes any real number and endpoint value within the numerical range, for example 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5, 1:10, 1:10.5, 1:11, 1:11.5, 1:12.

[0020] Further preferably, the resin bed diameter height ratio is 1:6-12; this ratio includes any real number and endpoint value within the numerical range, for example 1:6, 1:6.5, 1:7, 1:7.5, 1:7.7, 1:7.8, 1:7.9, 1:8, 1:8.1, 1:8.2, 1:8.3, 1:8.5, 1:9, 1:9.5, 1:10, 1:10.5, 1:11, 1:11.5, 1:12.

[0021] More preferably, the resin bed diameter-to-height ratio is 1:8.

[0022] In the present invention, the resin bed diameter-to-height ratio means: the ratio of the inner diameter of the macroporous resin column to the height of the filler after the macroporous resin column is filled.

[0023] Preferably, in step (1), the loading volume of the sample solution is 0.5-3BV; the loading volume includes any real number and endpoint value within the numerical range, for example 0.5BV, 1BV, 1.2BV, 1.5BV, 2BV, 2.5BV, 3BV. In a specific embodiment of the present invention, the loading volume is 3BV.

[0024] Preferably, in step (2), the volume concentration of the dilute ethanol solution is 10-20%; the volume concentration includes any real number and endpoint value within the numerical range, for example 10%, 10.2%, 10.5%, 10.7%, 11%, 11.5%, 12%.

[0025] Further preferably, the volume concentration of the dilute ethanol solution is 10%.

[0026] Preferably, in step (2), the dilute ethanol solution is used in an amount of 2-6 BV; the amount includes any real number and endpoint value within the numerical range, for example 2 BV, 2.5 BV, 3 BV, 3.5 BV, 4 BV, 4.5 BV, 5 BV, 5.5 BV, 6 BV.

[0027] Further preferably, the dilute ethanol solution is used in an amount of 2-4BV; the amount includes any real number and endpoint value within the numerical range, for example 2BV, 2.5BV, 3BV, 3.5BV, 4BV.

[0028] Further preferably, the dilute ethanol solution is used in an amount of 4 BV.

[0029] Preferably, in step (2), the flow rate of the impurity removal elution is 2-4 BV / h; the flow rate includes any real number and endpoint value within the numerical range, for example 2 BV / h, 2.5 BV / h, 3 BV / h, 3.5 BV / h, 4 BV / h.

[0030] Further preferably, the flow rate of the impurity removal elution is 3BV / h.

[0031] Preferably, in step (3), the concentrated ethanol solution has a volume concentration of 50-80%, which includes any real number and endpoint value within the numerical range, for example 50%, 55%, 60%, 65%, 70%, 75%, and 80%.

[0032] Further preferably, the concentrated ethanol solution has a volume concentration of 50-70%; the volume concentration includes any real number and endpoint value within the numerical range, for example 50%, 52%, 55%, 58%, 60%, 63%, 65%, 67%, and 70%.

[0033] More preferably, the concentrated ethanol solution has a volume concentration of 50%.

[0034] Preferably, in step (3), the elution flow rate is 2-4 BV / h; the flow rate includes any real number and endpoint value within the numerical range, for example 2 BV / h, 2.5 BV / h, 3 BV / h, 3.5 BV / h, 4 BV / h.

[0035] Further preferably, the elution flow rate is 4 BV / h.

[0036] Preferably, the concentrated ethanol solution is used in an amount of ≥4 BV; more preferably ≥5 BV; further preferably 5-10 BV; most preferably 5 BV.

[0037] Preferably, the drug is a drug that promotes polar wound healing and inhibits scar formation.

[0038] Further preferably, the drug is a drug that can improve the relative wound healing rate and the relative scar inhibition rate.

[0039] Further preferably, the drug is a drug that can increase the number of new capillaries, epidermal regeneration rate, the number of new skin appendages, collagen fiber ratio, type III collagen / type I collagen, and reduce the number of fibroblasts.

[0040] Further preferably, the drug is a drug that can reduce the levels of IL-6 and TNF-α in injured tissues, increase the level of VEGF-A in injured tissues, and increase the content of MMP3 protein.

[0041] Then, the present invention provides a pharmaceutical preparation, comprising the total phenolic acid of blue cloth obtained by the purification method described above.

[0042] Preferably, the types of the pharmaceutical preparation include gel, emulsion, ointment, cream, and patch.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] 1. The total phenolic acid of blue cloth in the application of the present invention is prepared by a specific purification method. By using a specific type of macroporous adsorption resin for adsorption, controlling the diameter-to-height ratio of the resin bed, the concentration of the sample solution, the flow rate of the sample solution, and the sample amount, controlling the type of solvent for impurities removal, the solvent concentration, the solvent flow rate, and the solvent dosage, and further controlling the type of solvent for elution, the solvent concentration, the solvent flow rate, and the solvent dosage, the adsorption rate, desorption rate, and transfer rate of the total phenolic acid of blue cloth are improved, and the mass fraction (content) of the total phenolic acid of blue cloth is improved. In addition, the purification method has high stability and good repeatability.

[0045] 2. The indigofera total phenolic acid obtained by the purification method of the present invention is applied to the preparation of a drug that promotes polar wound healing and inhibits scar formation. It can promote wound healing and inhibit scar formation in full-thickness resection model mice; the indigofera total phenolic acid can improve the relative wound healing rate and the relative scar inhibition rate, significantly increase collagen fibers, and promote wound healing; at the same time, the levels of inflammatory factors IL-6 and TNF-α are significantly reduced, which can reduce inflammatory reactions; and the VEGF-A level is significantly increased, creating a more favorable environment for skin growth; at 30 days, the indigofera total phenolic acid can significantly increase the content of MMP3, prevent excessive matrix deposition, and inhibit scar formation. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 is the leakage curve.

[0047] Figure 2 These are pictures of the wound conditions of the mouse full-thickness excision model on days 0, 3, 6, 9, 12, and 30 after modeling.

[0048] Figure 3 A is a bar graph comparing the relative healing rates of the full-thickness excision model of mice during the wound healing period (0th, 3rd, 6th, 9th, and 12th days after modeling); Figure 3 B is the result of the relative scar inhibition rate of the mouse full-thickness cortical excision model on the 30th day after modeling.

[0049] Figure 4 These are the results of the effect of total phenolic acid from blue cloth on the pathology of mouse skin wounds, A is HE staining (×10, 10 times magnification), B is Masson staining (×10, 10 times magnification), and C is Sirius red staining (×20, 20 times magnification; ×200, 200 times magnification).

[0050] Figure 5It is a statistical chart of pathological staining indicators, A is the number of new capillaries, B is the number of fibroblasts, C is the epidermal regeneration rate, D is the number of new skin appendages, E is the collagen fiber ratio, and F is type III collagen / type I collagen. DETAILED DESCRIPTION

[0051] The following non-limiting examples can enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way. The following content is merely an exemplary description of the scope of the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, which should also fall within the scope of the present invention.

[0052] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings as those generally understood by those of ordinary skill in the art to which the present invention belongs.

[0053] The present invention is further described below by way of specific examples. Unless otherwise specified, the various chemical reagents used in the examples of the present invention are obtained through conventional commercial channels.

[0054] In the following examples, the HPD-400 macroporous adsorption resin, HPD-600 macroporous adsorption resin, AB-8 macroporous adsorption resin, 101 macroporous adsorption resin and HPD-722 macroporous adsorption resin were purchased from Cangzhou Baoen Adsorption Material Technology Co., Ltd., with batch numbers of 20220822, 20220825, 20220829, 20220830 and 20220825. Products from different manufacturers have no significant effect on the effect.

[0055] The present invention tests the effects of the purification methods of the blue cloth total phenolic acid in each embodiment and comparative example, and investigates the adsorption rate, desorption rate, mass fraction (content), transfer rate and other aspects of the blue cloth total phenolic acid. The details are as follows:

[0056] (1) Mass fraction (content) of total phenolic acids in blue cloth

[0057] Preparation of reference solution: Accurately weigh 5 mg of gallic acid reference substance (batch number 110831-201906, purity 91.5%), place it in a 100 mL volumetric flask, ultrasonically dissolve it in distilled water, make up to volume, and shake well to obtain.

[0058] Determination of total phenolic acid content in blue cloth: Take the sample solution to be determined and dilute it appropriately, accurately measure 2mL and place it in a 25mL volumetric flask, accurately measure 1mL of phosphomolybdic tungstic acid test solution, 10mL of water, and 29% sodium carbonate solution to the scale, shake well, and let it stand for 30 minutes. Use the corresponding reagent as blank and measure the absorbance at a wavelength of 760nm.

[0059] The total phenolic acid content of the test sample was calculated by the absorbance ratio with that of the reference sample.

[0060] (2) Adsorption rate of total phenolic acids on blue cloth

[0061] The adsorption rate of total phenolic acid of blue cloth by macroporous adsorption resin: adsorption rate % = (C0V0-C1V1) / C0V0×100%;

[0062] Wherein, C0 is the concentration of total phenolic acid of blue cloth in the sample solution in mg / mL; C1 is the concentration of total phenolic acid of blue cloth in the sample solution flowing out after the adsorption operation in step (1) in mg / mL; V0 is the amount of sample solution (sample volume, mL); V1 is the amount of sample solution (sample volume, mL) flowing out after the adsorption operation in step (1).

[0063] (3) Desorption rate of total phenolic acids from blue cloth

[0064] The total phenolic acid in the blue cloth obtained after elution in step (3) was tested for content (C2) and volume (V2) was recorded, and the desorption rate was calculated using the following formula: desorption rate % = C2V2 / (C0V0-C1V1) × 100%;

[0065] Wherein, C0 is the concentration of total phenolic acid of blue cloth in the sample solution in mg / mL; C1 is the concentration of total phenolic acid of blue cloth in the sample solution flowing out after the adsorption operation in step (1) in mg / mL; C2 is the concentration of total phenolic acid of blue cloth obtained after elution in step (3) in mg / mL; V0 is the sample volume of the sample solution (sample volume, mL); V1 is the amount of the sample solution flowing out after the adsorption operation (volume, mL); V2 is the volume of total phenolic acid of blue cloth obtained after elution in step (3) in mL.

[0066] (4) Transfer rate of total phenolic acid in blue cloth

[0067] Transfer rate %=C2V2 / C0V0×100%;

[0068] Wherein, C0 is the concentration of total phenolic acid of blue cloth in the sample solution in mg / mL; C2 is the concentration of total phenolic acid of blue cloth obtained after elution in step (3) in mg / mL; V0 is the sample volume of the sample solution (sample volume, mL); V2 is the volume of total phenolic acid of blue cloth obtained after elution in step (3) in mL.

[0069] In the following embodiment, the preparation method of the indigofera extract comprises the steps of weighing indigofera, crushing, adding 15-25 times the amount of 50-60% ethanol, soaking at 50-60°C for 10-16h, filtering, concentrating the filtrate under reduced pressure at 40-60°C, and drying under reduced pressure to obtain the indigofera extract. Within the parameter range of the above preparation method, the indigofera extract used in this application can be obtained.

[0070] For example, in the embodiments and comparative examples of the present invention, the indigofera extract used is prepared by the method shown in Basic Example 1.

[0071] Basic Example 1

[0072] The preparation method of the indigofera extract comprises the following steps: weighing indigofera, crushing it, adding 20 times the amount of 55% ethanol, stirring and soaking it at 55°C for 12 hours, filtering, concentrating the filtrate under reduced pressure at 50°C, and drying it under reduced pressure at 60°C to obtain the indigofera extract.

[0073] Example 1

[0074] The method for purifying total phenolic acid of blue cloth comprises the following steps:

[0075] (1) Load the sample solution (the extract of blue bud extract was dissolved in water) with a concentration of 8 mg / mL at a flow rate of 3 BV / h into a HPD-400 macroporous adsorption resin column with a resin bed diameter-to-height ratio of 1:4-12, with a loading volume of 3 BV for adsorption;

[0076] (2) Impurity removal: Use a dilute ethanol solution with a volume concentration of 10% for impurity removal elution. The flow rate of the impurity removal elution is 3BV / h, and the amount of the dilute ethanol solution used for impurity removal elution is controlled to be 4BV;

[0077] (3) Elution: After impurities are removed, the macroporous adsorption resin is eluted with a concentrated ethanol solution with a volume concentration of 50%. The elution flow rate is controlled to be 4BV / h, and the amount of 50% concentrated ethanol solution is 5BV. After elution, the ethanol eluate is collected to obtain the total phenolic acid of blue cloth.

[0078] The results of the purification method of Example 1 are shown in Table 1:

[0079] Table 1

[0080]

[0081] The purification method of the invention has high adsorption rate, desorption rate and transfer rate for the total phenolic acid of indigofera, and the obtained total phenolic acid of indigofera has a high mass fraction.

[0082] Example 2

[0083] The difference from Example 1 is that the macroporous adsorption resin used in step (1) is different. The results of the purification method of this example are shown in Table 2.

[0084] Example 2.1: Replaced with HPD-600 macroporous adsorption resin;

[0085] Example 2.2: replaced with AB-8 macroporous adsorption resin;

[0086] Example 2.3: Replaced with D101 macroporous adsorption resin.

[0087] Comparative Example 1

[0088] The difference from Example 1 is that the macroporous adsorption resin used in step (1) is different. It is replaced by HPD-722 macroporous adsorption resin. The purification method effect results of this comparative example are shown in Table 2.

[0089] Table 2

[0090]

[0091] Example 3

[0092] Different from Example 1, the concentration of the sample solution in step (1) is 10 mg / mL and the sample volume is 2.4 BV. The results of the purification method are shown in Table 3.

[0093] Comparative Example 2

[0094] The difference from Example 1 is that the concentration of the loading solution in step (1) is 2 mg / mL and the loading volume is 12 BV. The results of the purification method are shown in Table 3.

[0095] Table 3

[0096]

[0097] Example 4

[0098] The difference from Example 1 is that the flow rate in step (1) is 3 BV / h. The results of the purification method are shown in Table 4.

[0099] Example 5

[0100] The difference from Example 1 is that the resin bed diameter-to-height ratio is different. The results of the purification method are shown in Table 4.

[0101] Example 5.1: Different from Example 1, the resin bed diameter-to-height ratio is 1:6.

[0102] Example 5.2: Different from Example 1, the resin bed diameter-to-height ratio is 1:12.

[0103] Example 5.3: Different from Example 1, the resin bed diameter-to-height ratio is 1:4.

[0104] Comparative Example 3

[0105] The difference from Example 1 is that the flow rate in step (1) is 4.5 BV / h. The results of the purification method are shown in Table 4.

[0106] Comparative Example 4

[0107] The difference from Example 1 is that the resin bed diameter-to-height ratio is 1:1. The results of the purification method are shown in Table 4.

[0108] Table 4

[0109]

[0110] Example 6

[0111] Different from Example 1, in step (1), the loading amount of the loading liquid is different. The loading amounts are 0.5BV, 1BV, 1.5BV, 2BV, 2.5BV, 3BV, 3.5BV, 4BV, 4.5BV and 5BV respectively. Then, after adsorption, the effluent is collected. The leakage curve is drawn with the concentration of total phenolic acid of blue cloth in each effluent and the initial injection mass concentration (C1 / C0) as the vertical coordinate and the flow fraction as the horizontal coordinate. Figure 1 As shown in the figure, 0.5 BV is one fraction, and the total phenolic acid content of blue cloth in each effluent is measured. When it reaches 10% of the initial injection mass concentration, it can be considered that there is leakage. When the sample volume is ≤3 BV (6 fractions), the ratio of total phenolic acid of blue cloth leaked is less than 10%.

[0112] Example 7

[0113] The difference from Example 1 is that the concentration of the dilute ethanol solution in step (2) is different, and the concentration of the dilute ethanol solution in this example is 20%. The results of the purification method are shown in Table 5.

[0114] Comparative Example 5

[0115] The difference from Example 1 is that the concentration of the dilute ethanol solution in step (2) is different. In this comparative example, the concentration of the dilute ethanol solution is 30%. The results of the purification method are shown in Table 5.

[0116] Table 5

[0117]

[0118] Example 8

[0119] The difference from Example 1 is that in step (2), the amount of the dilute ethanol solution used is different. The results of the purification method are shown in Table 6.

[0120] Example 8.1: The amount of dilute ethanol solution used in step (2) is 2 BV;

[0121] Example 8.2: The amount of dilute ethanol solution used in step (2) is 6 BV.

[0122] Comparative Example 6

[0123] The difference from Example 1 is that in step (2), the amount of the dilute ethanol solution used is different, 8 BV. The results of the purification method are shown in Table 6.

[0124] Table 6

[0125]

[0126] Example 9

[0127] The difference from Example 1 is that in step (2), the flow rate of the impurity removal elution is different. The results of the purification method are shown in Table 7.

[0128] Example 9.1: The flow rate of impurity removal elution is 2 BV / h;

[0129] Example 9.2: The flow rate of impurity removal elution is 4 BV / h.

[0130] Comparative Example 7

[0131] The difference from Example 1 is that in step (2), the flow rate of impurity removal elution is 6 BV / h. The results of the purification method are shown in Table 7.

[0132] Table 7

[0133]

[0134] Example 10

[0135] The difference from Example 1 is that the concentration of the concentrated ethanol solution used in step (3) is different. The results of the purification method are shown in Table 8.

[0136] Example 10.1: The concentration of the concentrated ethanol solution used in step (3) is 60%;

[0137] Example 10.2: The concentration of the concentrated ethanol solution used in step (3) is 80%;

[0138] Example 10.3: The concentration of the concentrated ethanol solution used in step (3) is 70%.

[0139] Comparative Example 8

[0140] The difference from Example 1 is that the concentration of the concentrated ethanol solution used in step (3) is 40%. The results of the purification method are shown in Table 8.

[0141] Table 8

[0142]

[0143] Embodiment 11

[0144] The difference from Example 1 is that the elution flow rate in step (3) is 2 BV / h. The results of the purification method are shown in Table 9.

[0145] Comparative Example 9

[0146] The difference from Example 1 is that the elution flow rate in step (3) is 7 BV / h. The results of the purification method are shown in Table 9.

[0147] Table 9

[0148]

[0149] Content detection of index ingredients

[0150] HPLC was used to determine the content of the index components in the tannin fraction of blue cloth. The chromatographic conditions were: Waters Sunfire C 18 Chromatographic column (250mm×4.6mm, 5μm); mobile phase: methanol (A)-0.1% formic acid water (B), gradient elution (0-8min, A 8%-8%; 8-25min, A 8%-20%; 25-45min, A 20%-40%; 45-55min, A 40%-55%; 55-65min, A 55%-90%); flow rate: 1.0mL / min; column temperature: 35℃; detection wavelength: 273nm; injection volume: 5μL.

[0151] Agilent Technologies 1200 Series high performance liquid chromatograph (Agilent, USA).

[0152] Preparation of test solution: Take the dried powder of the purified part of total phenolic acid of blue cloth, accurately weigh 50 mg, put it in a 10 mL volumetric flask, add methanol, ultrasonically dissolve, make up to volume, shake well, pass through a 0.22 μm microporous filter membrane, and take the filtrate to obtain the test solution.

[0153] Preparation of reference solution: Accurately weigh appropriate amounts of dimerized ellagitannin A (purity ≥ 95%), ethyl gallate (batch number 23010866, purity ≥ 98%, purchased from Beijing BetterKang Biotechnology Co., Ltd.), and ellagic acid reference substance (batch number 111959-201903, purity 88.8%, purchased from China Food and Drug Inspection Institutes), place them in a volumetric flask, make up to volume with methanol, and store at 4°C.

[0154] In the total phenolic acid of the blue cloth obtained in Example 1, Comparative Example 5 and Comparative Example 8, each index component was tested, and the content results (%) of each component are shown in Table 10.

[0155] Table 10

[0156]

[0157] Application Examples

[0158] 1. Grouping, establishment of mouse full-thickness cortical resection model, and drug administration

[0159] 168 SPF male ICR mice, weighing (25±2) g, were provided by Beijing Weitonglihua Experimental Animal Technology Co., Ltd., license number SCXK (Beijing) 2021-0006. They were kept in the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences. The temperature, humidity, and ventilation rate were automatically controlled by the central system. The temperature was maintained at 20-26°C, the relative humidity was maintained at 40%-70%, and the light was 12h light and 12h dark. All mice were adaptively fed 7 days before the experiment and had free access to food and water. The use of experimental animals was approved by the Experimental Animal Ethics Committee of the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, approval number: 2023B180.

[0160] 168 ICR mice were adaptively raised for 7 days and randomly divided into a model group, a recombinant human epidermal growth factor (rhEGF) group (0.1 g / cm 2 ) (recombinant human epidermal growth factor was purchased from Guilin Huanovi Gene Pharmaceutical Co., Ltd., batch number 202211067B), Example 1, low, medium and high (1.75 mg / mL, 3.5 mg / mL, 7 mg / mL) dose groups of total phenolic acid in blue cloth, comparative example 5, high dose group of total phenolic acid in blue cloth (7 mg / mL), comparative example 8, high dose group of total phenolic acid in blue cloth (7 mg / mL), all underwent full-thickness resection modeling. 1% sodium pentobarbital at 50 mg·kg -1 The mice were anesthetized with a dose of , and after anesthesia, the hair on the back of the mice was shaved with a shaver, and a full-thickness skin defect model was made on the back of the mice with the spine as the midline using a skin puncher, that is, two circular symmetrical holes with a diameter of about 10 mm, and the epidermis, dermis and subcutaneous tissue were removed to expose the meat fat membrane. The corresponding drug solution was applied to the wound in each treatment group, and the model group was applied with physiological saline solution. The drug was applied immediately after modeling, and 50 μL of the drug solution was drawn by a pipette to the wound, and evenly applied with a sterile cotton ball soaked with the drug solution. The drug was applied once every 24 hours for 30 consecutive days, during which SPF-grade animals were given regular feed and drinking water.

[0161] 2. Determination of relative wound healing rate and relative scar inhibition rate

[0162] The wound area of ​​each mouse was measured at 0, 3, 6, 9, 12, and 30 days after administration. The wound condition was recorded using a camera at a fixed height. The wound area was analyzed using ImageJ, and the area was measured after the image size was uniformed according to a ruler. The relative wound healing rate and relative scar inhibition rate were calculated using the following calculation formulas.

[0163] Relative wound healing rate = (Day 0 wound area - Day n wound area) / Day 0 wound area × 100%;

[0164] Relative scar inhibition rate = (Day 0 wound area - Day n scar area) / Day 0 wound area × 100%.

[0165] Figure 2 The wound conditions of the mouse full-thickness excision model on days 0, 3, 6, 9, 12, and 30 after modeling; Figure 2 Recorded in the figure are the model group, the rhEGF group, the low-dose group of total phenolic acid of indigofera in Example 1, the medium-dose group of total phenolic acid of indigofera in Example 1, and the high-dose group of total phenolic acid of indigofera in Example 1.

[0166] Figure 3 A is a bar graph comparing the relative healing rates of the full-thickness excision model of mice during the wound healing period (0th, 3rd, 6th, 9th, and 12th days after modeling); Figure 3 B shows the relative scar inhibition rate of the mouse full-thickness cortical excision model on the 30th day after modeling. Figure 3 Recorded in the figure are the model group, the rhEGF group, the low-dose group of total phenolic acid of indigofera in Example 1, the medium-dose group of total phenolic acid of indigofera in Example 1, and the high-dose group of total phenolic acid of indigofera in Example 1. Figure 3 Compared with the model group, # P<0.05, ## P<0.01.

[0167] Depend on Figure 2 and Figure 3 It can be seen that compared with the model group, the group treated with total phenolic acid from blue cloth (Example 1) significantly increased the wound healing rate at 3, 6, 9, and 12 days; at 30 days, the low, medium, and high dose groups of total phenolic acid from blue cloth in Example 1 significantly inhibited the scar area, indicating that total phenolic acid from blue cloth has the effect of promoting wound healing and inhibiting scar formation.

[0168] 3. Skin histopathology

[0169] Eight mice in each group were randomly killed at 6, 12, and 30 days after administration. The full-thickness skin tissue and a part of normal tissue were collected from the wound surface 2 mm along the wound edge. The left wound was collected and placed in 4% paraformaldehyde tissue fixative. The tissue was treated with formic acid formaldehyde decalcification solution, dehydrated with different concentrations of ethanol solution, transparentized with xylene, embedded in paraffin, and sectioned. The samples at 6, 12, and 30 days were stained with HE and Masson and observed under a microscope after sealing. The samples at 12 and 30 days were stained with Sirius red and observed under a polarized light microscope after sealing. The number of new capillaries, the number of fibroblasts, the number of skin appendages, the epidermal regeneration rate (regenerated epithelium length / total skin length), the collagen fiber ratio (collagen-positive blue area / total tissue area), and type III collagen / type I collagen (type III green collagen area / type I yellow collagen area) were counted by ImageJ. The results are shown in Figure 5. Figure 4 and Figure 5 (Recorded are the test results of the model group, rhEGF group, low-dose group of total phenolic acid of blue cloth in Example 1, medium-dose group of total phenolic acid of blue cloth in Example 1, and high-dose group of total phenolic acid of blue cloth in Example 1).

[0170] HE, Masson, and Sirius red staining were used to evaluate the wound healing and scar inhibition process at 6, 12, and 30 days. Figure 4 As shown in A, HE staining results showed that all groups had obvious inflammatory cell infiltration and exudate at 6 days, but the exudate in the rhEGF group and the low, medium and high dose groups of blue cloth total phenolic acid in Example 1 was significantly reduced. On 12 days, compared with the model group, the rhEGF group and the blue cloth total phenolic acid administration group in Example 1 had a relatively complete epidermis, and the inflammatory infiltration and exudate were significantly reduced. At 30 days, compared with the model group, the rhEGF group and the blue cloth total phenolic acid administration group in Example 1 grew more skin appendages such as hair follicles and sebaceous glands, indicating that the administration can promote wound healing and inhibit scar formation. The statistical results showed that after administration of rhEGF and total phenolic acid of blue cloth in Example 1, the number of new blood vessels increased significantly at 6d and 12d compared with the model group (P<0.05, 0.01), the number of fibroblasts decreased significantly (P<0.01), the epidermal regeneration rate was significantly higher than that of the model group (P<0.05, 0.01), and the number of new skin appendages increased significantly at 30d (P<0.01). Figure 5 .

[0171] like Figure 4 As shown in B, the Masson staining results showed that compared with the model group, the collagen fibers of Example 1 were significantly increased at 6, 12, and 30 days after the administration of total phenolic acid. Figure 5As shown in E, the statistical results showed that compared with the model group, the collagen fiber ratios of the low and high doses of total phenolic acid in Example 1 were significantly increased in the 6-day rhEGF group and the low and high doses of total phenolic acid in Example 1 (P<0.05, 0.01), and the collagen fiber ratios of the low, medium and high doses of total phenolic acid in the 30-day rhEGF group, Example 1 were significantly increased (P<0.01).

[0172] like Figure 4 As shown in C, the Sirius red staining results showed that at the 12-day wound growth stage, the rhEGF group and the low-, medium-, and high-dose groups of total phenolic acid in Example 1 had more type III collagen, indicating better skin healing effects. At 30 days, type III collagen decreased. The statistical results showed that at 12 days, the ratio of type III collagen to type I collagen in the rhEGF group and the total phenolic acid administration group in Example 1 increased significantly (P<0.01). At 30 days, only the high-dose group of total phenolic acid in Example 1 had a significant increase in the ratio of type III collagen to type I collagen (P<0.05). Figure 5 As shown in F.

[0173] 4. Enzyme-linked immunosorbent assay (ELISA) to detect the protein content of IL-6, TNF-α, VEGF-A, MMP3 and MMP9 in skin tissue

[0174] BCA protein quantification kit (Thermo scientific company, batch number 23225); mouse interleukin 6 (IL-6) ELISA kit, mouse matrix metalloproteinase 9 (MMP9) ELISA kit, mouse matrix metalloproteinase 3 (MMP3) ELISA kit, mouse high-sensitivity tumor necrosis factor α (TNF-α) ELISA kit, mouse vascular endothelial growth factor A (VEGF-A) ELISA kit (Wuhan Elaruite Biotechnology Co., Ltd., batch numbers E-EL-M0044c, E-EL-M3052, E-EL-M0626c, E-EL-M0009, E-EL-M1292c, respectively).

[0175] Eight mice in each group were randomly killed at 6, 12, and 30 days after administration. The full-thickness skin tissue and a portion of normal tissue were collected from the wound surface 2 mm along the wound edge. The skin tissue homogenate was prepared by adding physiological saline at a ratio of 1:9 and rotating at 3500 r / min below 4°C. -1After centrifugation for 10 min, the supernatant was taken and diluted, and the protein concentration was determined using a BCA protein quantification kit. The IL-6, TNF-α, VEGF-A, MMP3 and MMP9 protein contents in the skin tissue were detected strictly according to the instructions of the ELISA kit. The results are shown in Table 11.

[0176] Table 11

[0177]

[0178] In Table 11, compared with the model group, # P<0.05, ## P<0.01.

[0179] As shown in Table 11, compared with the model group, at 6 days, the levels of inflammatory factor IL-6 in the rhEGF group and the low-, medium- and high-dose groups of total phenolic acid of blue cloth in Example 1 were significantly reduced (P<0.01), and the IL-6 levels in the high-dose group of Comparative Example 5 and the high-dose group of Comparative Example 8 showed a downward trend, but there was no significant difference; the TNF-α level in the high-dose group of total phenolic acid of blue cloth in Example 1 was significantly reduced (P<0.05), and there was no significant difference in the NF-α level in the high-dose group of Comparative Example 5 and the high-dose group of Comparative Example 8; This indicates that the total phenolic acid of blue cloth in Example 1 has a significant anti-inflammatory effect in the acute stage of wounds, which is superior to Comparative Examples 5 and 8.

[0180] In the 12-day healing period, the levels of inflammatory factors (IL-6, TNF-α) in each administration group were not significantly different from those in the model group, indicating that the inflammatory level in the model group decreased during the healing and scar formation periods, and there was no significant difference between the administration groups; in the 12-day wound healing stage, the VEGF-A level in the rhEGF group and the low-dose group of total phenolic acid in Example 1 was significantly increased (P<0.05, 0.01), indicating that after the administration of total phenolic acid in Example 1, as the inflammation gradually eased, the VEGF-A level was promoted to increase, creating a more favorable environment for skin growth. At 12 days, the MMP3 level in the rhEGF group and the medium- and high-dose groups of total phenolic acid in Example 1 was significantly reduced (P<0.05, 0.01), and the MMP9 level in the rhEGF group and the high-dose group of total phenolic acid in Example 1 was significantly reduced (P<0.05, 0.01). The above results indicate that total phenolic acid in Example 1 significantly inhibited the activity of MMPs during the healing period, which helped to reduce excessive tissue degradation and maintain the integrity of the matrix.

[0181] At 30 days of scar formation, there was no significant difference in the levels of inflammatory factors (TNF-α) in each drug-treated group compared with the model group, indicating that the inflammatory level in the model group decreased during the scar formation period and was not significantly different from that in the drug-treated group; at 30 days, the MMP3 level in the high-dose group of total phenolic acid from blue cloth in Example 1 was significantly increased (P<0.05), and the MMP9 level in the rhEGF group was significantly increased (P<0.05), indicating that during the scar formation period, total phenolic acid from blue cloth in Example 1 significantly increased the activity of MMP3, which helped prevent excessive matrix deposition and scar formation.

[0182] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. An application of total phenolic acid of blue cloth in the preparation of a drug for promoting wound healing and inhibiting scar formation, characterized in that: Total phenolic acids of blue cloth are obtained by a purification method comprising the following steps: (1) Loading a sample solution containing a blue cloth extract onto a macroporous adsorption resin column having a resin bed diameter-to-height ratio of 1:4-12 at a flow rate of 2-4 BV / h for adsorption; the sample solution is an aqueous solution of the blue cloth extract at a concentration of 4-10 mg / mL; and the loading volume of the sample solution is 0.5-3 BV; The preparation method of the indigofera extract comprises the following steps: weighing indigofera, crushing it, adding 15-25 times the amount of 50-60% ethanol, soaking it at 50-60° C. for 10-16 hours, filtering it, concentrating the filtrate under reduced pressure at 40-60° C., and drying it under reduced pressure to obtain the indigofera extract; (2) Impurity removal: Use a dilute ethanol solution with a volume concentration of 5-20% for impurity removal elution. The flow rate of the impurity removal elution is 2-4BV / h, and the amount of the dilute ethanol solution for impurity removal elution is controlled to be 2-6BV; (3) Elution: Use a concentrated ethanol solution with a volume concentration of 50-80% for elution, control the elution flow rate to 2-6BV / h, collect the ethanol eluate, and obtain the total phenolic acid of blue cloth.

2. The use according to claim 1, characterized in that: In step (1), the macroporous adsorption resin is selected from one of HPD-400 macroporous adsorption resin, HPD-600 macroporous adsorption resin, AB-8 macroporous adsorption resin and D101 macroporous adsorption resin.

3. The use according to claim 1, characterized in that: In step (2), the volume concentration of the dilute ethanol solution is 10-20%.

4. The use according to claim 3, characterized in that: The dilute ethanol solution has a dosage of 2-4 BV; the volume concentration of the dilute ethanol solution is 10%; and the flow rate of the impurity removal elution is 3 BV / h.

5. The use according to claim 1, characterized in that: In step (3), the elution flow rate is 2-4 BV / h.

6. The use according to claim 1, characterized in that: The medicine is a medicine capable of improving the relative wound healing rate and the relative scar inhibition rate.

7. The use according to claim 1, characterized in that: The drug is a drug that can increase the number of new capillaries, epidermal regeneration rate, the number of new skin appendages, collagen fiber ratio, type III collagen / type I collagen, and reduce the number of fibroblasts.

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