Dandelion emplastrum as well as preparation method and application thereof

Abstract

The invention discloses dandelion emplastrum as well as a preparation method and application thereof, and belongs to the technical field of biological extracts. The invention provides a dandelion extract, and a preparation method of the dandelion extract comprises the following steps: drying whole dandelion, soaking in distilled water for 0.5 h, heating twice, 2 h each time to obtain a water extract, and removing moisture to obtain the dandelion extract. The mass ratio of the whole dandelion herb to the distilled water is 1: 20. The moisture removal is that the water extract is dried by distillation in a water bath and then is dried at a constant temperature of 60 DEG C. The dandelion is prepared into the emplastrum, and the emplastrum has a good repairing effect when being used for repairing abdominal skin permeability and wound surfaces of rats. A scientific basis is provided for further development and research of dandelion external preparations, and a clinical basis is conveniently provided for research on treatment of local trauma.

Description

Technical Field

[0001] The present invention relates to the field of biological extracts, and particularly to a dandelion plaster and its preparation method and application. Background Art

[0002] Dandelion (Taraxacum mongolicum Hand.-Mazz.) is a perennial plant that commonly grows in areas such as hillsides, grasslands, roadsides, fields, and riverbanks. It is the dried whole herb of the dandelion plant of the Compositae family. Mongolian dandelion, also known as dandelion, has the Mongolian name Bagebaigai-Qiqiqige and the Tibetan name Kurimang. In Mongolian medicine, it has the effects of calming "Xieri", clearing heat, detoxifying, and promoting appetite. Although the development of products and preparations of dandelion at home and abroad has been continuously evolving, there is currently no dandelion external preparation on the market.

[0003] Wound repair is a complex pathological process. Wound repair refers to a complex process in which proliferating cells and extracellular matrix regenerate, proliferate, fill, and connect after injury. It is a process in which multiple cells assist each other in wound healing. The process of skin wound healing is mainly divided into four stages: coagulation stage, inflammation stage, proliferation stage, and remodeling stage, and the four stages overlap with each other. Wounds are external skin injuries caused by various factors. If disinfection and antibacterial treatment are not carried out, they will be damaged in an indescribable way. Dandelion is one of the traditional medications and has high medical value. Although the development of products and preparations of dandelion at home and abroad has been continuously evolving, there is currently no dandelion external preparation on the market. Summary of the Invention

[0004] The purpose of the present invention is to provide a dandelion plaster and its preparation method and application to solve the problems existing in the above-mentioned prior art. The present invention makes a dandelion extract into a plaster, which shows good repair effects on the skin penetration rate and wound repair of the rat abdomen.

[0005] To achieve the above purpose, the present invention provides the following solutions:

[0006] Technical Solution 1: A dandelion dry extract, and the preparation method of the dandelion dry extract includes the following steps: After drying the whole dandelion herb, soak it in distilled water for 0.5 h and heat it twice, each time for 2 h, to obtain a water extract, and obtain the dandelion dry extract after evaporating the water.

[0007] Further, the mass ratio of the whole dandelion herb to distilled water is 1:20.

[0008] Further, the evaporation of water is to evaporate the water extract by water bath and then dry it at a constant temperature of 60 °C.

[0009] Technical Solution 2: A dandelion plaster for treating skin trauma, the active ingredient including the dandelion dry extract described above.

[0010] Furthermore, the components of the dandelion plaster include the dandelion dry extract and auxiliary materials; the auxiliary materials are glycerol.

[0011] Furthermore, the mass ratio of the dandelion dry extract to the glycerol is 1:0.5.

[0012] Technical Solution 3: Application of the dandelion dry extract described above in the preparation of a drug for skin wound repair.

[0013] Furthermore, the skin wound repair includes increasing the skin wound healing rate and promoting the formation of new capillaries and granulation tissue.

[0014] The present invention discloses the following technical effects:

[0015] The present invention takes Taraxacum mongolicum Hand.-Mazz. as the research object, screens out the optimal process for water decoction extraction, prepares the dandelion plaster and measures its content and content uniformity. After evaluating its permeation performance through in vitro percutaneous penetration experiments, the wound repair effect is studied. The dandelion plaster prepared by the present invention is in the coagulation stage on the first day of administration. It can be seen that small blood vessels, capillaries, etc. near the wound contract, resulting in a decrease in blood flow and the formation of clots to achieve the effect of coagulation and hemostasis. The 7th to 14th days of administration are the inflammation stage and the proliferation stage. It can be seen from the result graph that a large number of inflammatory cells migrate and proliferate at the wound site and then slowly decrease. After that, the keratinocytes become actively proliferating, resulting in the formation of granulation tissue and the reconstruction of a new vascular network on the wound surface. On the 18th day, that is, the remodeling stage, the granulation tissue completely covers the wound and a large number of hair follicles begin to appear. This process will continue for some time until the wound is completely covered with hair. From the above, it can be concluded that the dandelion plaster prepared by the present invention has an obvious promoting effect on the repair of rat skin trauma. Moreover, the dandelion plaster prepared by the present invention has good adhesiveness and holding force, and the drug release degree is moderate. Patients can apply the drug by themselves, which is convenient and practical. It can avoid the first-pass effect of the liver that may occur during oral administration and reduce the side effects of the gastrointestinal tract. In short, the present invention makes the water decoction of dandelion into a plaster, which shows good repair effect on the percutaneous permeability and wound repair of rat abdominal skin. It provides a scientific basis for the further development of the research on dandelion topical preparations and is convenient for providing a clinical basis for the future research on the treatment of local trauma. Description of the Drawings

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0017] Figure 1 is the standard curve of chlorogenic acid;

[0018] Figure 2 is the picture of the manual plaster;

[0019] Figure 3 is the linear relationship diagram of chlorogenic acid;

[0020] Figure 4 is the penetration content results at different time points;

[0021] Figure 5 is the wound healing situation of each treatment group at different time periods;

[0022] Figure 6 is the HE staining result diagram of each treatment group at different time periods, and the magnification is 100 times. Specific implementation manners

[0023] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0024] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0025] Example 1

[0026] 1. Dandelion water decoction extraction process

[0027] The reference substance chlorogenic acid (batch number AZ22011851, purity ≥ 98%) was provided by Tianjin Yongda Chemical Reagent Co., Ltd.; phosphoric acid (batch number 20191110) was purchased from Tianjin Yongda Chemical Reagent Co., Ltd. The experimental medicinal material dandelion was identified by Professor Bao Guihua of the School of Mongolian Medicine, Inner Mongolia University for Nationalities as the dried whole herb of Taraxacum mongolicum Hand.-Mazz. of the genus Taraxacum in the Compositae family, and was collected from Jinmei Jiayuan, Tongliao City, Inner Mongolia.

[0028] 2 Methods and results

[0029] 2.1 Preparation method of dandelion extract

[0030] Take 9 portions of dried dandelion whole herb, each portion being 50.0 g, place them in a 1000 mL round-bottom flask, add 10, 15, and 20 times the mass of distilled water respectively, soak for 30 minutes and then heat. Use strong fire (220 V) before boiling and gentle fire (90 V) after boiling, decoct for 1, 1.5, and 2.0 h respectively, and decoct 1, 2, and 3 times respectively to obtain 9 portions of water extract extracts. Load them into an evaporating dish, evaporate to dryness in a water bath (80 °C) and then continue to dry at a constant temperature of 60 °C in a drying oven to obtain dry extracts. The orthogonal experimental design is shown in Table 1.

[0031] Table 1

[0032]

[0033] 2.2 Preparation of test solution

[0034] Precisely weigh 0.16 g of the dried dandelion water extract, place it in a conical flask, add 10 mL of methanol with a volume percentage of 20%, shake well and let it stand at room temperature for 10 min, filter with filter paper, and then use a filter membrane to filter. The obtained dandelion water extract solution is the test solution.

[0035] 2.3 Preparation of reference substance

[0036] Precisely weigh 1 mg of chlorogenic acid reference substance and place it in a 5 mL volumetric flask, add methanol to the scale and dissolve. Prepare a chlorogenic acid reference solution with a concentration of 0.2 mg / mL for standby.

[0037] 2.4 Chromatographic conditions

[0038] Chromatographic column: Waters Atlantis T3 chromatographic column (4.6×250 mm, 5 μm); Mobile phase: acetonitrile - 0.4% phosphoric acid aqueous solution; Injection volume: 5 μL; Flow rate: 1 mL / min; Column temperature: 25 °C; Wavelength: at 323 nm. The gradient elution ratio is shown in Table 2.

[0039] Table 2

[0040] Time (min) Mobile phase A (%) Mobile phase B (%) 0.01 12 88 20 18 82

[0041] 2.5 Investigation of linear relationship

[0042] Precisely absorb 2.5 mL, 5.0 mL, 7.5 mL, 10.0 mL, and 12.5 mL of the reference solution under item "2.4", set the injection volume as 5 μL, and determine the peak area value by HPLC under the above chromatographic conditions. The injection volume of the reference substance is the abscissa, and the peak area A(y) of the reference substance is the ordinate. Then, through linear regression, the chlorogenic acid standard curve equation is: y = 442.32x + 93.542, and the correlation coefficient r = 0.9995; The results show that 0.5 - 2.5 mg·mL -1There is a good linear relationship between the product value of chlorogenic acid and the peak area within a certain range, as Figure 1 shown.

[0043] 2.6 Precision experiment investigation

[0044] Precisely pipette 5 μL of each reference substance stock solution, inject it into the liquid chromatograph 6 times, measure and record the peak area, statistically calculate its RSD%, and obtain 1.4%, indicating that the precision of the instrument is good under this condition. The results are shown in Table 3.

[0045] Table 3

[0046]

[0047] 2.7 Repeatability experiment investigation

[0048] Take sample Y9, prepare 6 portions in parallel according to the determination method of the test solution, perform HPLC analysis under the chromatographic conditions of "2.4", with an injection volume of 5 μL, record the peak area of its chromatographic peak, calculate RSD%, and through analysis, its RSD% is 1.8%, indicating that the repeatability of this extraction method is good. The results are shown in Table 4.

[0049] Table 4

[0050]

[0051] 2.8 Stability experiment investigation

[0052] Take the sample of the ninth batch, prepare the test solution according to the preparation method of the test solution, perform HPLC analysis under the chromatographic conditions of "2.4", inject and measure at 0, 2, 4, 8, 12, and 24 h respectively, with an injection volume of 5 μL. The results show that the RSD of the peak area of chlorogenic acid is 1.3%. The experimental results show that the test sample is stable within 24 h. The results are shown in Table 5.

[0053] Table 5

[0054]

[0055] 2.9 Spiked recovery experiment investigation

[0056] Take 0.08 g of the sample with known content, a total of 6 portions, add chlorogenic acid reference substance according to the requirement that the ratio of the known amount in the sample to the added amount of the reference substance is 1:1, prepare the test solution according to the preparation method of the test solution under item "2.2", perform HPLC analysis under the chromatographic conditions of "2.4". The average recovery rate of chlorogenic acid is 102.25%, and the RSD is 1.8%. The results are shown in Table 6.

[0057] Table 6

[0058]

[0059] 2.10 Investigation Results of Dandelion Extraction Process

[0060] When water is used as the extraction solvent, the main influencing factors are the amount of added water, decoction time, and number of decoction times. Therefore, three levels are determined for each factor to design an orthogonal experiment. The results of the orthogonal experiment are shown in Table 7 (Orthogonal Experiment Design and Results Table of L9(34)) and Table 8 (Analysis of Variance Results). It can be seen from the results that the optimal extraction method is A3B3C2, and the order of influencing factors is A > B > C.

[0061] Table 7

[0062]

[0063] Table 8

[0064]

[0065]

[0066] Taking the content of chlorogenic acid and the extraction rate of dandelion water decoction as indexes, the factors and conditions affecting the extraction of chlorogenic acid were screened through orthogonal experiments, and the best extraction process was obtained as follows: the amount of added water was 20 times that of the original medicinal materials, the decoction time was 2 h, and the number of decoction times was 2 times.

[0067] Example 2

[0068] Preparation of Dandelion Plaster

[0069] 1. Experimental Reagents

[0070] The reagents include ultrapure water and glycerol (20210701) purchased from Tianjin Damao Chemical Reagent Factory

[0071] 2. Methods and Results

[0072] 2.1 Liquid Medicine Extraction Process

[0073] According to the orthogonal design results of Example 1, after cleaning and soaking the dried whole dandelion herb for 0.5 h, add 20 times the amount of water and decoct for 2 times. Each time after 2 h, the obtained dandelion water decoction is concentrated and reserved on a water bath.

[0074] 2.2 Selection and Standard of the Ratio of Main Drug to Auxiliary Materials

[0075] According to the obtained paste rate, convert the dose of the plaster (the dose of raw medicinal powder for external use on the skin recorded in the "Technical Specification for Clinical External Use of Traditional Chinese Medicine Powders" is 1 - 3 g / cm 2) In this case, the dosage of the main drug (dandelion extract) is 1 g. By reducing the dosage ratio of glycerol, the fluidity, solubility, and stability of the solution were investigated. Taking the presence or absence of particles in the solution, whether the ointment and glycerol were evenly dissolved, and whether the ointment was stable on the adhesive tape as the criteria, that is, a brown solution, the solution did not separate, the drug did not sink, the color of the ointment was uniform, and the ointment did not fall off to meet the standard.

[0076] When adding glycerol, the mixing property was weak and the fluidity was too strong at a ratio of 1:1.5; the mixing stability of dandelion extract and glycerol was weak and the fluidity was good at a ratio of 1:1; when the ratio was 1:0.5, the solubility, mixing property, and fluidity were all stronger than the above two ratios. Therefore, the ratio of dandelion extract to excipients was selected as 1:0.5. See Table 9 for details.

[0077] Table 9

[0078] Active ingredient: Excipient (g: g) Comprehensive evaluation 1:1.5 Strong fluidity, incomplete dissolution with particles, and not firmly adhered to the tape 1:1 Good fluidity, with a small amount of particles during incomplete dissolution, not firmly adhered to the tape 1:0.5 Good fluidity, complete dissolution without particles, firmly adhered to the tape

[0079] 2.3 Manual preparation of the patch

[0080] Preparation of the main drug: According to the results of the orthogonal experiment in Example 1, the dried whole dandelion herb was cleaned, soaked for 0.5 h, then added with 20 times the amount of water and decocted twice, each time for 2 h. The combined decoction was evaporated and concentrated in a water bath and then dried in an oven to obtain a dry extract, which was pulverized and reserved. Mixing of the drug and excipients: The experimental prescription amount was dandelion water decoction extract powder: excipients = 1 g: 0.5 g. That is, the dandelion water decocted dry extract powder was mixed with glycerol at a ratio of 1:0.5 and stirred to obtain the ointment. Laying the gauze: In the "Technical Specification for Clinical External Use of Traditional Chinese Medicine Powders", the dose of raw drugs for external use on the skin is 1 - 3 g / cm 2 , so the dose of the patch was converted according to the extract yield of the above experiment. The converted amount was about 0.35 g / cm 2 , the ointment was applied to the gauze to obtain the medicated gauze. The medicated gauze was placed on the medical adhesive tape, and after complete adhesion, it was wrapped with a protective paper film and then cut to manually make a simple dandelion patch. See the picture of the manual patch in Figure 2 .

[0081] Example 3

[0082] Determination of the content and content uniformity of the dandelion patch

[0083] 1. Experimental materials

[0084] Reference substance chlorogenic acid (purity ≥ 98%, batch number AZ22011851), reference substance rutin (batch number AZ21080201, purity ≥ 98%) were both provided by Chengdu Efa Biotechnology Co., Ltd.; chromatographic pure methanol (batch number 20220402), analytical pure methanol (batch number 20220505), phosphoric acid (batch number 20191110) were all purchased from Tianjin Yongda Chemical Reagent Co., Ltd.

[0085] 2. Methods and Results

[0086] 2.1. Preparation of Solutions

[0087] 2.1.1 Preparation of Reference Substances

[0088] Precisely weigh 1.16 mg of rutin and 1.0 mg of chlorogenic acid, add methanol to a 5 mL volumetric flask to make up the volume. The concentration of rutin was configured to 0.232 mg·mL -1 The concentration of chlorogenic acid was configured to 0.2 mg·mL -1 solution.

[0089] 2.1.2 Preparation of Test Solution

[0090] Weigh the dandelion ointment, add 50% methanol, let it stand for 10 minutes and filter, and filter through a 0.22 μm filter membrane pore to obtain the test solution.

[0091] 2.2 Chromatographic Conditions

[0092] Agilent ZORBAX Eclipse Plus C 18 chromatographic column (250 nm × 4.6 nm, 5 μm); using methanol (A): 0.4% phosphoric acid water (B) as the mobile phase, the injection volume was 10 μL; flow rate: 0.7 mL / min; column temperature was 20 °C; wavelength was at 323 nm. The gradient elution ratio is shown in Table 10.

[0093] Table 10 Chromatographic Conditions

[0094]

[0095]

[0096] 2.3 Methodological Verification

[0097] 2.3.1 Investigation of Linear Relationship

[0098] Inject 5.0 μL, 7.5 μL, 10.0 μL, 12.5 μL, and 15 μL of the reference solution under item "2.1.1", and perform HPLC determination and analysis according to the chromatographic conditions in "2.2" to calculate the peak area values. Take the injection volume of the reference solution as the abscissa and the peak area A(y) as the ordinate for linear regression. The standard curve equation of chlorogenic acid is: y = 1225.5x - 472.23, and the correlation coefficient r = 0.9995; the results show that the linear relationship of chlorogenic acid is good in the range of 1.16 - 3.48 mg·mL -1 The linear relationship of rutin is good in the range of 1 - 3 mg·mL -1 range.

[0099] 2.3.2 Precision experiment investigation

[0100] Inject the prepared chlorogenic acid reference solution and rutin into the instrument 6 times repeatedly according to the above chromatographic conditions, record their peak areas, calculate RSD%, and obtain that the RSD of chlorogenic acid is 1.5% and that of rutin is 1.35%, indicating that the instrument precision is good under this condition.

[0101] 2.3.3 Repeatability experiment investigation

[0102] Weigh the same sample (weigh 6 portions), prepare it according to the preparation method of the test sample, perform HPLC analysis under the chromatographic conditions, record the peak areas of the chromatographic peaks simultaneously, and calculate RSD%. The results show that the RSD% are 1.5% (chlorogenic acid) and 1.8% (rutin) respectively, indicating that the repeatability of this extraction method is good. The results are shown in Table 11.

[0103] Table 11

[0104]

[0105] 2.3.4 Stability experiment investigation

[0106] Take an appropriate amount of the sample, prepare the test sample solution according to the preparation method of the test sample solution, analyze it under the chromatographic conditions, and inject and determine it at 0, 4, 8, 10, 12, and 24 h respectively. The results show that the RSD of the peak area of chlorogenic acid is 1.5% and the RSD of the peak area of rutin is 1.2%, indicating that the test sample is stable within 24 h.

[0107] 2.3.5 Spiked recovery test investigation

[0108] Take 6 samples of the same batch, accurately weigh them, place them in a conical flask after accurate weighing, add appropriate amounts of the reference substances chlorogenic acid and rutin, prepare the test solution according to the preparation method of the test solution, inject and analyze according to the above chromatographic conditions, measure and record, calculate the recovery rate and RSD value of the components therein. The calculated average recovery rate of chlorogenic acid is 99.5%, and the RSD is 1.6%. The average recovery rate of rutin is 101.93%, and the RSD is 1.1%. The results are shown in Table 12.

[0109] Table 12

[0110]

[0111] 2.4 Determination of content uniformity

[0112] Take 10 tablets of each of 3 batches of samples, grind them separately and place them in a 10 mL volumetric flask, add 10 mL of 50% methanol to volume to the mark, shake well, filter, and use it as the content uniformity solution. Check according to the law of content uniformity in General Chapter 0941 of the Chinese Pharmacopoeia (Part IV) 2020 Edition, and calculate the average content of 10 tablets, the absolute value A of the difference between the labeled amount and the mean value, the standard deviation S, and the content uniformity (n = 10, A + 2.2S) value. The results are shown in Table 13.

[0113] Table 13

[0114]

[0115]

[0116] The present invention is applicable to experiments using two brands of chromatographic columns, namely, Agilent ZORBAX Eclipse Plus C18 chromatographic column (250 nm × 4.6 nm, 5 μm) and Waters Atlantis T3 chromatographic column (4.6 × 250 mm, 5 μm). By comparing the separation degree of chromatographic peaks, the chemical information measured, and the peak shape of the peaks to be measured; finally, the present invention is applicable to the Agilent ZORBAX Eclipse Plus C18 chromatographic column (250 nm × 4.6 nm, 5 μm) chromatographic column.

[0117] For the content determination experiment, gradient elution was performed using two solvents: methanol-phosphoric acid water and acetonitrile-phosphoric acid water. When the mobile phase was methanol (A) - 0.4% phosphoric acid water (B), the elution sequence was as follows: from 0 to 5 minutes, 10% - 18% A; from 5 to 20 minutes, 18% - 45% A; from 20 to 24 minutes, 45% - 48% A; from 24 to 30 minutes, 48% - 60% A. In this case, there was more chemical information and better separation effect. In addition, the column temperature and injection volume were also investigated in this invention. The results showed that when the column temperature was 20 °C, 25 °C, and 30 °C, there was no influence on the resolution of the chromatographic peaks. Therefore, considering the actual peak shape, a column temperature of 25 °C was selected, and when the injection volume was 10 microliters, the effect was the best. According to the content uniformity inspection method in the General Principles 0941 of the Chinese Pharmacopoeia (Volume IV, 2020 Edition), the content uniformity of three batches of cataplasms was within the standard range. A + 2.2S ≤ L (L ≤ 25.0). The experimental results showed that the linear relationship of the reference substance was good. From the results of the stability, repeatability, and precision experiments, the stability, repeatability, and precision of this method were good, and the RSD% was less than 2%. This indicated that the experimental conditions and methods of this invention were reliable, providing a safe and reliable method for the content determination of dandelion cataplasm.

[0118] Example 4

[0119] Study on Percutaneous Penetration and Wound Repair Effects of Cataplasm in Vitro

[0120] 1. Experimental Materials

[0121] 1.1 Experimental Animals and Equipment

[0122] SD rats (half male and half female) with a body weight of 200 ± 20 g were purchased from Liaoning Changsheng Biotechnology Co., Ltd. (License No.: SCXK (Liao) 2022 - 00001). The experimental animals were fed in the animal room of the School of Mongolian Medicine, with free access to food and water, and the temperature was between 22 and 25 °C.

[0123] 1.2 Main Instruments and Materials

[0124] The instruments are shown in Table 14, and the reagents and materials are shown in Table 15.

[0125] Table 14

[0126]

[0127]

[0128] Table 15

[0129]

[0130] 2 Experimental Methods

[0131] 2.1 Preparation of Ex Vivo Rat Skin

[0132] Take healthy SD rats weighing about 200±20 g. After abdominal anesthesia with chloral hydrate, use a hair clipper to remove all the hair on the rat's abdomen, and then carefully remove the remaining hair tissue with a razor. After sacrificing the treated rats, immediately remove the abdominal skin with surgical scissors. Lay the removed skin flat on a smooth wooden board and carefully remove the subcutaneous fat tissue and connective tissue with surgical scissors, taking care to ensure that the skin is not damaged. Finally, rinse the treated rat skin thoroughly with normal saline and place it in a sealed bag for storage at -80°C for later use. Before the experiment, check again to ensure that the skin is not damaged.

[0133] 2.2 In vitro percutaneous penetration experiment

[0134] Use the Ussing chamber experimental device (effective diffusion area 0.5 cm 2 ), to investigate the in vitro percutaneous permeability of dandelion plaster. After thawing the excised skin, wash it once carefully with normal saline and wipe off the moisture with filter paper. Stick a certain area of the band-aid on the stratum corneum side of the skin, with the inner side of the skin facing the receiving pool, and fix it between the fixing clips. Adjust the water bath temperature to (37±0.5°C), and the receiving medium is normal saline. Take 2 ml of samples at 0.5 h, 1 h, 2 h, 4 h, 6 h, and 8 h respectively and replenish an equal amount and same-temperature medium in a timely manner. Filter the samples with a filter membrane and then analyze them by HPLC injection, and record their peak areas.

[0135] 2.3 HPLC methodology investigation

[0136] 2.3.1 Preparation of reference substance

[0137] Accurately weigh 1.00 mg of chlorogenic acid and place it in a volumetric flask (5 ml), add methanol to dissolve it. Prepare a solution with a reference substance concentration of 0.2 mg / ml for standby.

[0138] 2.3.2 Preparation of samples

[0139] Filter the receiving liquid solutions at different time periods with a microporous filter membrane and then analyze them by HPLC injection.

[0140] 2.3.3 Chromatographic conditions

[0141] Chromatographic column: T3 chromatographic column (250 mm×4.6 mm, 5 μm); Mobile phase: acetonitrile - 0.4% phosphoric acid aqueous solution (87; 13); Flow rate: 1 ml / min; Column temperature: 25°C; Detection wavelength: 327 nm; Injection volume: 10 μL. Under these conditions, the peak emergence time of chlorogenic acid is about 9 - 10 min, and the peak shape is good.

[0142] 2.3.4 Investigation of linear relationship

[0143] Precisely aspirate 5.0 mL, 7.5 mL, 10.0 mL, 12.5 mL, and 15 mL of the reference solution under item "3.2", inject the samples, and determine their peak area values under the above chromatographic conditions. Take the injection volume of the reference solution as the abscissa and the peak area A(y) as the ordinate for linear regression to obtain the standard curve equation of chlorogenic acid: y = 124.26x + 891.96, with a correlation coefficient r = 0.9995. The results show that within the range of 0.5 - 2.5 mg·mL -1 the linear relationship of the reference chlorogenic acid is good, as shown in Figure 3 .

[0144] 2.3.5 Precision experiment investigation

[0145] Inject the prepared chlorogenic acid (0.2 mg / mL) reference solution continuously for 6 times, record the peak area, calculate the RSD%, and obtain 2%, indicating good precision under this condition. The results of the precision experiment are shown in Table 16.

[0146] Table 16

[0147]

[0148]

[0149] 2.3.6 Stability experiment investigation

[0150] Take the receiving solution at the same time period, inject samples at 0, 2, 4, 8, 12, and 24 h respectively to determine the peak area. The relative standard deviation is 1.1%, indicating good stability of the receiving solution within 24 h. The results of the stability experiment are shown in Table 17.

[0151] Table 17

[0152]

[0153] 2.3.7 Repeatability experiment investigation

[0154] Select 6 sample solutions at the same time period, analyze them under the above chromatographic conditions, record the peak area of the chromatographic peak simultaneously, calculate the RSD%. The result of RSD% is 1.9%, indicating good repeatability of the extraction method. The results of the repeatability experiment are shown in Table 18.

[0155] Table 18

[0156]

[0157] 2.3.8 Spike recovery test investigation

[0158] An appropriate amount of chlorogenic acid reference substance was accurately weighed and added to the sample with a known concentration, and then analyzed by injection under the chromatographic conditions to determine its peak area. The average recovery rate of chlorogenic acid was 102.79%, and the RSD was 1.75%.

[0159] 3. Research on wound repair effect

[0160] 3.1 Administration and grouping of experimental animals

[0161] The rats raised for seven days were randomly divided into 6 groups, namely the normal group, the model group (without administration), the positive control group (Yunnan Baiyao plaster), the low-dose (0.1 g / kg) administration group, the medium-dose (0.2 g / kg) administration group, and the high-dose (0.3 g / kg) administration group, with 24 rats in each group, and they were raised individually in cages.

[0162] 3.2 Establishment of wound model

[0163] One day before modeling, the backs of the rats in each group were shaved with a hair remover, and they were not restricted from drinking water or eating. The next day, after the rats were anesthetized by intraperitoneal injection of chloral hydrate, circular skin wounds were artificially created on the backs of the rats in the model group and other groups except the normal group, and the wound depth was uniformly controlled to the depth of the fascia layer. According to the relevant literature method, 1 hour after the modeling was completed, when the wound slightly shrank, the drugs were administered. The positive group was administered Yunnan Baiyao plaster, and the model group was not administered. The back was wrapped with medical tape for one week and the rats were raised individually in cages, and the dressing was changed daily until complete healing.

[0164] 3.3 Preparation of samples to be tested

[0165] After modeling, the wounds were photographed and recorded on days 1, 7, 14, and 18 (the photographing angle and height should be kept consistent), and the wound area was measured using ImageJ image software. The obtained area data were used to calculate the healing rate of each group according to the formula: (wound area on day 1 - wound area on day n) / wound area on day 1 × 100%. At the same time, on days 1, 7, 14, and 18, after anesthesia with 10% chloral hydrate, the back wound tissues were taken and fixed in 10% formaldehyde solution for preparing pathological sections. The data were expressed as mean ± standard deviation and analyzed using SPSS 26 data analysis software. P < 0.05 was considered to indicate a statistically significant difference.

[0166] 3.4 Detection of pathological indicators

[0167] 3.4.1 Pathological sections

[0168] ① Gradient dehydration: After taking out the tissues with forceps from 10% formaldehyde solution and washing away the excess impurities with distilled water, each group of tissues was trimmed and placed in an embedding cassette, and left overnight in 75% ethanol. The next day, they were soaked in 80% ethanol, 95% ethanol I, 95% ethanol II, absolute ethanol I, and absolute ethanol II for 1 h, 1.5 h, 1.5 h, 1 h, and 1 h respectively.

[0169] ② Clearing: The embedding cassette was moved to xylene I and xylene II and soaked for 10 min respectively.

[0170] ③ Wax infiltration: The embedding cassette was successively placed into preheated wax baths I and II, with each wax infiltration for 20 min and the temperature being 65 °C each time.

[0171] ④ Embedding: After wax infiltration, the skin tissue was picked up with forceps, placed in the embedding cassette, and embedded by dropping wax (first fill the bottom of the embedding cassette with paraffin and gently place the tissue in it). After covering the embedding cassette, it was transferred to a low temperature for cooling and stored overnight in a 4 °C refrigerator. The temperature of the embedding table was optimally 65 °C.

[0172] ⑤ Sectioning, spreading, and picking up sections: After separating the wax block from the embedding cassette, it was placed on the fixed clamp of the microtome, and the wax block was sectioned into slices with a thickness of 4 μm. The wax slices were gently pinched with tissue forceps and a writing brush and carefully placed into a constant temperature water bath at 37 °C. Let the sections float on the water surface and then gently unfold the sections with forceps, and pick up the sections onto the glass slides.

[0173] ⑥ Baking the slides: The glass slides were fixed on a fixed rack and placed in an oven for 3 h for standby.

[0174] 3.4.2 HE staining

[0175] ① Dewaxing to water: The glass slides were successively placed into environment-friendly dewaxing solution I and environment-friendly dewaxing solution II for dewaxing, with each time being 20 min; then the glass slides were hydrated with ethanol from high concentration to low concentration, successively being absolute ethanol I (5 min), absolute ethanol II (5 min), 75% alcohol (5 min), and finally rinsed with running water.

[0176] ② Hematoxylin staining: The glass slides were stained in hematoxylin staining solution for 3 - 5 min, rinsed with tap water; differentiated with the differentiating solution; then the glass slides were rinsed with tap water again.

[0177] ③ Blueing: After blueing in the blueing solution, rinsed with running water; dehydrated in 85% and 95% gradient alcohols for 5 min each.

[0178] ④ Eosin staining: The glass slides were placed in eosin staining solution and stained for 5 min.

[0179] ⑤Dehydration and mounting: The glass slides were soaked in ethanol with different concentrations for dehydration, namely absolute ethanol I, absolute ethanol II, absolute ethanol III, xylene I, and xylene II, each for 5 minutes for transparency.

[0180] ⑥Mounting: After the glass slides were dried, they were mounted with neutral balsam.

[0181] 4. Statistical analysis method

[0182] In this invention, IBM SPSS Statistics 26 statistical analysis software was used for data statistical analysis and processing. The data were expressed as mean ± standard deviation and P < 0.05 was considered statistically significant.

[0183] 5. Experimental results

[0184] 5.1 Results of the content of chlorogenic acid in the receiving solution at different time points

[0185] The receiving solutions sampled at 0.5 h, 1 h, 2 h, 4 h, 6 h, and 8 h in these 6 different time periods were respectively processed and their contents were determined under the above chromatographic conditions. The permeation content results at different time points are shown in Table 19 and Figure 4 as follows.

[0186] Table 19

[0187] Time 0.5h 1h 2h 4h 6h 8h Content 0％ 0.18％ 0.64％ 0.56％ 0.45％ 0.43％

[0188] It can be seen from the linear graph that in the in vitro percutaneous permeation rate experiment, the content of chlorogenic acid was zero at 0.5 h, indicating that the drug was in the stage of adhering to the skin and releasing the drug at this time; after 1 h, it permeated into the skin to play a therapeutic effect, reached the highest concentration at 2 h, and exerted its best pharmacodynamic effect, and the content was 0.64% at this time. Then it gradually decreased, but remained at a relatively high content. It began to level off at a relatively high content at 6 h, indicating that it could play a therapeutic effect for a long time.

[0189] 5.2 Macroscopic observation results of each group of rats at different time points

[0190] All animals were in good general condition postoperatively, without any adverse reactions and with normal activities. From the wound morphology and wound healing rate, it could be seen that on the day of model establishment, all rat wounds had bleeding and edema, and after a period of time, blood clots formed and filled the wounds. On the 1st day after model establishment, the wounds of all rats had a certain degree of contraction, but the difference was minimal. There was a small amount of purulent substance in the model group, and a small amount of blood could be seen on the wound surface of some rats. The wound surfaces of the other four groups were dry, and there were no statistical differences among the groups (P > 0.05). On the 7th day, the wound healing degrees of each group were different. Scar formation occurred in the low-dose drug administration group, positive control group, and high-dose group, and the wound healing was obvious. It was observed that the model group was ruddy and accompanied by a small amount of bleeding, which was speculated to be caused by external infection leading to inflammation. A white and smooth skin appeared around the wound surfaces of each group, which was a manifestation after wound healing. There was no statistical difference between the medium-dose and high-dose drug administration groups and the model group (P > 0.05), and there was a statistical difference between the high-dose drug administration group and the positive control group (P < 0.05). On the 14th day, the wound healing was obvious. The wounds of the low-dose drug administration group and the positive control group were basically healed, presenting linearly. The pinkish new skin could be clearly seen at the wound edges, and the hair around began to regrow. The scabs of all rats' wounds in the medium-dose and high-dose drug administration groups had completely fallen off, and new skin tissues began to grow around. Scar formation began in the model group and slowly fell off. There was a statistical difference between the low-dose drug administration group and the medium- and high-dose drug administration groups (P < 0.05), no statistical difference between the medium-dose and high-dose drug administration groups (P > 0.05), and no statistical difference between the low-dose drug administration group and the positive control group (P > 0.05). On the 18th day, the positive control group and the low-dose drug administration group were completely healed without scars, and the newly grown hair completely covered the wounds, with a healing rate reaching 98%. New skin tissues grew around the wounds of the model group and the high-dose drug administration group. There was no statistical difference between the low-dose drug administration group and the positive control group (P > 0.05), and there was a statistical difference between the low-dose drug administration group and the model group (P < 0.05). The wound healing rate is shown in Table 20 and Figure 5 as shown.

[0191] Table 20

[0192]

[0193] Note: ※ indicates a statistical difference compared with the model group (P < 0.05).

[0194] 5.3 HE staining results

[0195] From the HE staining results, it could be obtained that under the microscope (10×10) on the 1st day after drug administration, it was observed that small blood vessels, capillaries, etc. near the wounds of rats in each group contracted, resulting in a decrease in blood flow and the formation of blood clots, and inflammatory cells formed. The HE staining result diagrams of each treatment group at different time periods are shown in Figure 6On the 7th day of administration, the inflammatory cell infiltration in the model group was relatively obvious, and the number of fibroblasts and new blood vessels increased compared with before; a large number of new blood vessels and fibroblasts were visible in the low-dose administration group and the positive administration group, and a small amount of inflammatory cell infiltration was observed as granulation tissue began to form; under microscopic observation, the growth of new capillaries in the medium-dose administration group and the high-dose administration group was less than that in the low-dose administration group. On the 14th day, fibroblasts were obvious in the model group, and granulation tissue began to form; keratinocytes in the low-dose administration group and the positive administration group began to work, the round wound became elongated, inflammatory cells gradually decreased, a new blood vessel network was reconstructed, and a large amount of granulation tissue formed. A small amount of hair follicle tissue could be seen. The inflammatory cells in the medium-dose administration group and the high-dose administration group decreased significantly, and granulation tissue formation was obvious. On the 18th day, a large amount of granulation tissue formed in the model group, and keratinocytes began to work, and the round wound became elongated. There were more hair follicles in the low-dose administration group and the positive administration group, and the granulation tissue transformed into scar tissue and entered the tissue remodeling stage. Although hair follicles and a large number of granulation cells were generated in the medium-dose administration group and the high-dose administration group.

[0196] The embodiments described above are only used to describe the preferred mode of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A dried dandelion extract, characterized in that, The preparation method of the dandelion dry extract comprises the following steps: After drying the whole dandelion herb, soak it in distilled water for 0.5 h and heat it twice, 2 h each time, to obtain a water extract, and obtain the dandelion dry extract after evaporating the water.

2. The dandelion dry extract according to claim 1, wherein, The mass ratio of the whole dandelion herb to distilled water is 1:

20.

3. The dandelion dry extract according to claim 1, characterized in that, The evaporation of water is to evaporate the water extract in a water bath at 80 °C until dry, and then continue to dry it at a constant temperature of 60 °C to obtain a dry extract.

4. A dandelion plaster for treating skin trauma, characterized in that, The active ingredient comprises the dandelion dry extract described in any one of claims 1-3.

5. The dandelion plaster according to claim 4, wherein The components of the dandelion plaster include the dandelion dry extract and auxiliary materials; the auxiliary material is glycerol.

6. The dandelion plaster according to claim 5, characterized in that, The mass ratio of the dandelion dry extract to the glycerol is 1:0.

5.

7. Use of the dandelion dry extract described in any one of claims 1-3 in the preparation of a drug for skin wound repair.

8. The application according to claim 7, characterized in that, The skin wound repair includes increasing the skin wound healing rate and promoting the formation of new capillaries and granulation tissue.

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