A composition of effective parts of Chinese hawthorn and a preparation method thereof

Abstract

The present application relates to a kind of Huahong composition effective part extract and its preparation method, the Huahong composition effective part extract includes the following components: syringic acid, new chlorogenic acid, deacetyl carlina acid methyl ester, geniposidic acid, N-acetyl-L-tyrosine, fraxidin, 4-pyridoxinic acid, 7,8-dihydroxy-4-methyl coumarin, chlorogenic acid, ferulic acid;The total content of above components is 80%-95% of effective part extract.The Huahong composition effective part extract of the present application is from the effective part of Huahong composition.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to an effective extract of a safflower composition and its preparation method. Background Technology

[0002] The Huahong composition, represented by Huahong tablets, consists of the following medicinal ingredients: *Hedyotis diffusa*, *Hedyotis diffusa*, *Spatholobus suberectus*, *Rhodomyrtus tomentosa* root, *Bai Bei Ye* root, *Gnaphalium affine*, and *Iris tectorum*. Xiaozhong Zhitong Ding is a traditional Chinese medicine product of Guangxi Zhuang Autonomous Region Huahong Pharmaceutical Group Co., Ltd. It clears heat and detoxifies, dries dampness and stops leukorrhea, removes blood stasis and relieves pain. It is used for leukorrhea and irregular menstruation caused by damp-heat stagnation, characterized by excessive, yellow, thick leukorrhea, lower abdominal pain, lumbosacral pain, and menstrual cramps; it is also used for chronic pelvic inflammatory disease and adnexitis with the above symptoms.

[0003] The effective component of a traditional Chinese medicine (TCM) refers to a mixture of one or more chemical components in a single herb or compound herb extract, where the content of one or more chemical components reaches 50% or more of the total extract, and these one or more known chemical components are considered effective ingredients. New TCM drugs made from such extracts are called new drugs based on their effective components.

[0004] Research on the effective components of traditional Chinese medicine has many advantages, including conforming to traditional Chinese medicine theory and modern theories of "multi-component, multi-target, and multi-effect," making it an effective research approach for modernizing traditional Chinese medicine. Therefore, research on the effective components of safflower compositions is of great significance. Summary of the Invention

[0005] The purpose of this invention is to provide an extract of the effective components of a safflower composition and a method for preparing the same, wherein the extract is derived from the effective components of the safflower composition.

[0006] The effective fraction extract of the safflower composition comprises the following components:

[0007] Syringic acid, neochlorogenic acid, deacetylated methyl methacrylate, genipin, N-acetyl-L-tyrosine, fraxin, 4-pyridoxine, 7,8-dihydroxy-4-methylcoumarin, chlorogenic acid, ferulic acid;

[0008] The total content of the above components is 80%-95% of the effective fraction extract.

[0009] The weight parts of each component in the effective fraction extract are as follows:

[0010] Syringic acid 15-30 parts, neochlorogenic acid 200-300 parts, deacetylated methyl ester 8-20 parts, genipin 10-25 parts, N-acetyl-L-tyrosine 8-20 parts, fraxin 40-60 parts, 4-pyridoxine 35-55 parts, 7,8-dihydroxy-4-methylcoumarin 180-250 parts, chlorogenic acid 200-300 parts, ferulic acid 20-40 parts.

[0011] The aforementioned safflower composition is made from the following medicinal materials: *Hedyotis diffusa*, ...Spatholobus suberectus*, *Rhodomyrtus tomentosa* root, *Bai Bei Ye* root, *Gardenia jasminoides*, and *Illicium verum*.

[0012] The aforementioned safflower composition is made from the following medicinal materials in parts by weight:

[0013] 400-600 parts of *Hedyotis diffusa*, 200-400 parts of *Hedyotis diffusa*, 300-500 parts of *Spatholobus suberectus*, 400-600 parts of *Rhodomyrtus tomentosa* root, 200-400 parts of *Bai Bei Ye* root, 400-600 parts of *Ground Peach Blossom*, and 200-400 parts of *Illicium verum*.

[0014] The preparation method of the effective fraction extract of the aforementioned safflower composition includes the following steps:

[0015] Take the safflower extract, dilute it with water by 0.8-3 times, centrifuge, take the supernatant, pass it through a C18 column, let it stand for 4-8 hours, and then elute it sequentially with water equivalent to 20-40 times the weight of the supernatant and methanol equivalent to 20-40 times the weight of the supernatant by volume percentage. Collect the 20% methanol eluent, and dry it under vacuum to obtain the final product.

[0016] The method for preparing the safflower composition paste includes the following steps:

[0017] The ingredients, including *Hedyotis diffusa*, ...Spatholobus suberectus*, *Rhodomyrtus tomentosa* root, *Bai Bei Ye* root, *Gardenia jasminoides*, and *Illicium verum*, are extracted with water or alcohol and then concentrated into a thick paste.

[0018] The method for preparing the safflower composition paste includes the following steps:

[0019] Take all the medicinal materials, add water equivalent to 6-12 times the weight of all the medicinal materials, and decoct 2-3 times, 1-3 hours each time. Combine the decoctions, filter, and concentrate the filtrate to a clear extract with a relative density of 1.05-1.25 at 80℃. After cooling to room temperature, add ethanol to make the alcohol content 50-80%, stir well, let stand for 12-48 hours, and filter. Recover the ethanol from the filtrate to obtain an extract with a relative density of 1.20-1.30 at 80℃.

[0020] Another type of extract of the effective components of safflower composition is prepared by the following steps:

[0021] Take 8%-13% of the *Illicium verum* and grind it into a fine powder for later use; add the remaining *Illicium verum* and all other medicinal materials to water equivalent to 8-12 times their weight and decoct 2-3 times, 1-3 hours each time. Combine the decoctions, filter, and concentrate to a clear extract with a relative density of 1.15-1.25 at 80℃; after cooling to room temperature, add ethanol to make the alcohol content 65%, stir well, let stand for 24 hours, and filter; recover the ethanol from the filtrate to an extract with a relative density of 1.2-1.32 at 80℃, add the *Illicium verum* fine powder and mix well to obtain a mixture;

[0022] Take the mixture, dilute it with water by 0.8-3 times, centrifuge, take the supernatant, pass it through a C18 column, let it stand for 4-8 hours, then elute it sequentially with water (20-40 times the weight of the supernatant) and methanol (20-40 times the weight of the supernatant by volume percentage), collect the 20% methanol eluent, and dry it under vacuum to obtain the final product.

[0023] The present invention also discloses the use of the above-mentioned safflower composition effective fraction extract in the preparation of anti-inflammatory drugs.

[0024] Experiments have shown that the composition of this invention is non-toxic to zebrafish at doses of 0-500 μg / ml, and its anti-inflammatory effect is dose-dependent; the higher the concentration, the more significant the inhibition of neutrophil migration and the stronger the anti-inflammatory activity. Attached Figure Description

[0025] Figure 1 These are HPLC analysis chromatograms of the separated components of the sample prepared in Example 4;

[0026] Figure 2 This is an example of the migration of neutrophils in a zebrafish inflammation model based on the sample prepared in Example 4;

[0027] Figure 3 The effects of different concentrations of safflower extracts on inflammation in zebrafish were compared with those of the model group: **P<0.01, ****P<0.0001;

[0028] Figure 4 The effects of extracts of the effective components of different concentrations of safflower composition on reducing inflammation in zebrafish were compared with those of the model group: **P<0.01, ****P<0.0001;

[0029] Figure 5 The inhibitory rate of neutrophils on zebrafish inflammation model by extracts of the effective components of different concentrations of safflower composition. Detailed Implementation

[0030] The present invention is further illustrated below by way of examples. It should be understood that the examples of the present invention are for illustrative purposes and not for limiting the present invention. Simple modifications made to the present invention based on the essence of the present invention are all within the scope of protection claimed by the present invention.

[0031] Example 1

[0032] The effective fraction extract of the safflower composition comprises the following components:

[0033] The effective fraction extract of the safflower composition comprises the following components:

[0034] Syringic acid, neochlorogenic acid, deacetylated methyl methacrylate, genipin, N-acetyl-L-tyrosine, fraxin, 4-pyridoxine, 7,8-dihydroxy-4-methylcoumarin, chlorogenic acid, ferulic acid;

[0035] The total content of the above components is 80% of the effective fraction extract.

[0036] The relative contents of each component in the effective fraction extract are as follows:

[0037] Syringic acid 15mg / g, neochlorogenic acid 200mg / g, deacetylated methyl ester 8mg / g, genipin 10mg / g, N-acetyl-L-tyrosine 8mg / g, fraxin 40mg / g, 4-pyridoxine 35mg / g, 7,8-dihydroxy-4-methylcoumarin 180mg / g, chlorogenic acid 200mg / g, ferulic acid 20mg / g.

[0038] The preparation method of the effective component extract of the safflower composition includes the following steps:

[0039] Take the safflower composition paste, dilute it with water by 0.8 times, centrifuge, take the supernatant, pass it through a C18 chromatographic column, let it stand for 4 hours, and then elute it sequentially with water equivalent to 20 times the weight of the supernatant and methanol equivalent to 20 times the weight of the supernatant by volume percentage. Collect the 20% methanol eluent, and dry it under vacuum to obtain the final product.

[0040] The aforementioned safflower composition is made from the following medicinal materials in parts by weight:

[0041] 4kg of *Hedyotis diffusa*, 2kg of *Hedyotis diffusa*, 3kg of *Spatholobus suberectus*, 4kg of *Myrtle* root, 2kg of *White-backed Leaf* root, 4kg of *Gnaphalium affine*, and 2kg of *Ipomoea aquatica*.

[0042] The method for preparing the safflower composition paste includes the following steps:

[0043] Take all the medicinal materials, add water equivalent to 6 times the weight of all the medicinal materials and decoct twice, 1 hour each time. Combine the decoctions, filter, and concentrate the filtrate to a clear extract with a relative density of 1.05 at 80℃. After cooling to room temperature, add ethanol to make the alcohol content 50%, stir well, let stand for 12 hours, and filter. Recover the ethanol from the filtrate to obtain an extract with a relative density of 1.20 at 80℃.

[0044] Example 2

[0045] The effective fraction extract of the safflower composition comprises the following components:

[0046] Syringic acid, neochlorogenic acid, deacetylated methyl methacrylate, genipin, N-acetyl-L-tyrosine, fraxin, 4-pyridoxine, 7,8-dihydroxy-4-methylcoumarin, chlorogenic acid, ferulic acid;

[0047] The total content of the above components is 95% of the effective fraction extract.

[0048] The relative contents of each component in the effective fraction extract are as follows:

[0049] Syringic acid 30mg / g, neochlorogenic acid 300mg / g, deacetylated methyl ester 20mg / g, genipin glycoside 25mg / g, N-acetyl-L-tyrosine 20mg / g, fraxin 60mg / g, 4-pyridoxine 55mg / g, 7,8-dihydroxy-4-methylcoumarin 250mg / g, chlorogenic acid 300mg / g, ferulic acid 40mg / g.

[0050] The preparation method of the effective component extract of the safflower composition includes the following steps:

[0051] Take the safflower extract, dilute it with water three times, centrifuge, collect the supernatant, pass it through a C18 column, let it stand for 8 hours, and then elute it sequentially with water equivalent to 40 times the weight of the supernatant and methanol equivalent to 40 times the weight of the supernatant by volume percentage 20%. Collect the 20% methanol eluent, and dry it under vacuum to obtain the final product.

[0052] The aforementioned safflower composition is made from the following medicinal materials in parts by weight:

[0053] 6kg of *Hedyotis diffusa*, 4kg of *Hedyotis diffusa*, 5kg of *Spatholobus suberectus*, 6kg of *Myrtle Root*, 4kg of *White-backed Leaf Root*, 6kg of *Gardenia jasminoides*, and 4kg of *Ipomoea aquatica*.

[0054] The method for preparing the safflower composition paste includes the following steps:

[0055] Take all the medicinal materials, add water equivalent to 12 times the weight of all the medicinal materials and decoct 3 times, 3 hours each time. Combine the decoctions, filter, and concentrate the filtrate to a clear extract with a relative density of 1.25 at 80℃. After cooling to room temperature, add ethanol to make the alcohol content 80%, stir well, let stand for 48 hours, and filter. Recover the ethanol from the filtrate to obtain an extract with a relative density of 1.30 at 80℃.

[0056] Example 3

[0057] The effective fraction extract of the safflower composition comprises the following components:

[0058] Syringic acid, neochlorogenic acid, deacetylated methyl methacrylate, genipin, N-acetyl-L-tyrosine, fraxin, 4-pyridoxine, 7,8-dihydroxy-4-methylcoumarin, chlorogenic acid, ferulic acid;

[0059] The total content of the above components is 87% of the effective fraction extract.

[0060] The relative contents of each component in the effective fraction extract are as follows:

[0061] Syringic acid 20 mg / g, neochlorogenic acid 250 mg / g, deacetylated methyl ester 12 mg / g, genipin 13 mg / g, N-acetyl-L-tyrosine 11 mg / g, fraxin 52 mg / g, 4-pyridoxine 43 mg / g, 7,8-dihydroxy-4-methylcoumarin 210 mg / g, chlorogenic acid 240 mg / g, ferulic acid 30 mg / g.

[0062] The preparation method of the effective component extract of the safflower composition includes the following steps:

[0063] Take the safflower extract, dilute it with water by 1.2 times, centrifuge, take the supernatant, pass it through a C18 column, let it stand for 6 hours, and then elute it sequentially with water equivalent to 30 times the weight of the supernatant and methanol equivalent to 30 times the weight of the supernatant by volume percentage 20%. Collect the 20% methanol eluent, and dry it under vacuum to obtain the final product.

[0064] The aforementioned safflower composition is made from the following medicinal materials in parts by weight:

[0065] 5kg of *Hedyotis diffusa*, 3kg of *Hedyotis diffusa*, 4kg of *Spatholobus suberectus*, 5kg of *Myrtle* root, 3kg of *White-backed Leaf* root, 5kg of *Gnaphalium affine*, and 3kg of *Ipomoea aquatica*.

[0066] The method for preparing the safflower composition paste includes the following steps:

[0067] Take all the medicinal materials, add water equivalent to 8 times the weight of all the medicinal materials and decoct twice, 2 hours each time. Combine the decoctions, filter, and concentrate the filtrate to a clear extract with a relative density of 1.15 at 80℃. After cooling to room temperature, add ethanol to make the alcohol content 70%, stir well, let stand for 36 hours, and filter. Recover the ethanol from the filtrate to obtain an extract with a relative density of 1.25 at 80℃.

[0068] Example 4

[0069] The effective fraction extract of the safflower composition comprises the following components:

[0070] Syringic acid, neochlorogenic acid, deacetylated methyl methacrylate, genipin, N-acetyl-L-tyrosine, fraxin, 4-pyridoxine, 7,8-dihydroxy-4-methylcoumarin, chlorogenic acid, ferulic acid;

[0071] The total content of the above components is 80%-95% of the effective fraction extract.

[0072] The relative contents of each component in the effective fraction extract are as follows:

[0073] Syringic acid 23 mg / g, neochlorogenic acid 255 mg / g, deacetylated methyl ester 13 mg / g, genipin 17 mg / g, N-acetyl-L-tyrosine 13 mg / g, fraxin 48 mg / g, 4-pyridoxine 45 mg / g, 7,8-dihydroxy-4-methylcoumarin 219 mg / g, chlorogenic acid 242 mg / g, ferulic acid 27 mg / g.

[0074] The preparation method of the effective fraction extract of the aforementioned safflower composition includes the following steps:

[0075] Take the safflower extract, dilute it with water by 1:1, centrifuge, take 1.5 ml of the supernatant, pass it through a C18 column, let it stand for 5 hours, then use 50 ml of water and 50 ml of 20% methanol by volume in sequence, collect the 20% methanol eluent, and dry it under vacuum to obtain the final product.

[0076] The aforementioned safflower composition is made from the following medicinal materials in parts by weight:

[0077] 12.5kg of *Hedyotis diffusa*, 7.5kg of *Hedyotis diffusa*, 10kg of *Spatholobus suberectus*, 12.5kg of *Myrtle Root*, 7.5kg of *White-backed Leaf Root*, 12.5kg of *Gnaphalium affine*, and 7.5kg of *Ipomoea aquatica*.

[0078] The method for preparing the safflower composition paste includes the following steps:

[0079] Take all the medicinal materials, add water equivalent to 10 times the weight of all the medicinal materials and decoct twice, 2 hours each time. Combine the decoctions, filter, and concentrate the filtrate to a clear extract with a relative density of 1.20 at 80℃. After cooling to room temperature, add ethanol to make the alcohol content 65%, stir well, let stand for 24 hours, and filter. Recover the ethanol from the filtrate to obtain an extract with a relative density of 1.28 at 80℃.

[0080] Example 5

[0081] Chemical composition identification of extracts from effective parts of safflower composition

[0082] I. Sample Preparation

[0083] The extract of the effective fraction of the safflower composition prepared in Example 4 was dissolved in pure water as a solvent and filtered through a 0.22 μm microporous membrane to prepare a test solution with a concentration of 5 mg / ml.

[0084] II. Chromatographic Conditions

[0085] Chromatographic column: water XTERRA@RP18 (3.0×150mm, 3.5μm);

[0086] The mobile phase is 0.1% formic acid (A) and methanol (B);

[0087] Gradient elution: 0-18 min 90% A-76% A, 18-23 min 76% A, 23-60 min 76% A-50% A;

[0088] Detection wavelength: 326nm, column temperature: 25℃, flow rate: 0.6ml / min, injection volume: 5μl.

[0089] III. Mass Spectrometry Conditions

[0090] Mass spectrometry conditions: electrospray ionization source (ESI), positive and negative ion modes, scan range m / z 100~1000; collision energy 35e V; ion transfer tube temperature 320℃; vaporizer temperature 350℃; positive and negative ion spray voltages 3500 and 3000 V, respectively.

[0091] IV. Data Analysis

[0092] The analysis was performed using Compound Discoverer 3.0 software, matching analysis was conducted on five databases: Predicted Compositions, mzCloudSearch, mzVault Search, ChemSpider Search, and MassList Search, and then filtering and matching retrieval analysis were performed based on the ppm value range [-5 to +5].

[0093] V. Experimental Results

[0094] The effective fraction extract of the safflower composition was identified by liquid chromatography-mass spectrometry (LC-MS). Detection was performed under the aforementioned chromatographic and mass spectrometric conditions. The high-performance liquid chromatogram is shown below. Figure 1 As shown. Based on polarity, the peak times separated 10 components from the effective fraction extract of the safflower composition, namely: Syringic acid, Neochlorogenic acid, Deacetylasperulosidic acid methyl ester, Geniposidic acid, N-Acetyl-L-tyrosine, Esculin, 4-Pyridoxic acid, 7,8-Dihydroxy-4-methylcoumarin, Chlorogenic acid, and Ferulic acid.

[0095] Based on the relative peak area calculations, the relative contents of the active components of the safflower composition extract were as follows: Syringic acid 23 mg / g, Neochlorogenic acid 255 mg / g, Deacetylasperulosidic acid methyl ester 13 mg / g, Geniposidic acid 17 mg / g, N-Acetyl-L-tyrosine 13 mg / g, Esculin 48 mg / g, 4-Pyridoxic acid 45 mg / g, 7,8-Dihydroxy-4-methylcoumarin 219 mg / g, Chlorogenic acid 242 mg / g, and Ferulic acid... The relative content of ferulic acid is 27 mg / g.

[0096] Example 5

[0097] Anti-inflammatory experiments of the effective component extract of safflower compound based on a zebrafish inflammation model

[0098] I. Zebrafish strains and feeding management

[0099] Husbandry conditions: The equipment used is a UPU series ultrapure water system and a PENTAIR zebrafish aquatic ecosystem from the USA. The bottom is disinfected with ultraviolet light for 24 hours, and the fish room is kept at a constant temperature. The water temperature is 28-30℃, the salinity is 0.03%-0.04%, the pH is 7.2-7.6, the light cycle is 14:10 (14 hours of light followed by 10 hours of darkness), the lights are on from 8:30 am to 10:00 pm daily, and the fish are fed twice a day.

[0100] 2.3.2 Collection of Zebrafish Embryos

[0101] Adult male and female zebrafish were selected each evening and placed in aquariums with perforated bottoms, with a male-to-female ratio of 1:1. The following morning, after exposure to sunlight, the zebrafish mated and laid eggs. The eggs were collected promptly, and the zebrafish were returned to the aquarium. The collected eggs were then placed in 100mm culture dishes containing embryo culture water. The eggs were placed in a constant temperature incubator at 28.5±0.5℃ and cultured, with the water changed every 24 hours.

[0102] II. Establishment and Administration of a Zebrafish Embryo Immersion Copper Sulfate Inflammation Model

[0103] A zebrafish inflammation model was established by immersing fluorescent, healthy 3dpf zebrafish embryos in copper sulfate solution. The 3dpf zebrafish embryos were randomly divided into groups of 30 each.

[0104] (1) Control group: Zebrafish embryos were soaked in embryo culture water for 2 hours.

[0105] (2) Model group: Zebrafish embryos were immersed in embryo culture water with 20 μM copper sulfate for 2 h.

[0106] (3) Positive group: Zebrafish embryos were immersed in embryo culture water containing 20 μM copper sulfate and 10 μg / ml dexamethasone for 2 h.

[0107] (4) Test drug group: Zebrafish embryos were immersed in embryo culture water containing 125, 250, and 500 μg / ml of the effective part extract of the safflower composition prepared in Example 4, which contained 20 μM copper sulfate, for 2 h.

[0108] III. Effects of the components of the safflower paste on inflammatory factor-related mRNA in inflammatory zebrafish

[0109] The zebrafish were induced and administered the drug according to method "2.3.3". After culturing in an incubator at 28.5℃ for 2 hours, the zebrafish were washed three times with PBS. Total RNA was extracted from the zebrafish using the conventional trizol extraction method, and the RNA concentration was determined. Following the ReverTra... cDNA was obtained by reverse transcription using the qPCRRT Kit. The reverse transcription conditions were as follows: Step 1: 65℃ for 5 min; 4℃; remove, add reagents according to the instructions, then continue with Step 2: 37℃ for 15 min; 98℃ for 5 min; 4℃ to finish. The UDG SYBR qPCR SuperMix Plus kit instructions for Real-time PCR are as follows: Step 1: Pre-denaturation at 50°C for 120 s, one cycle; Step 2: PCR reaction at 94°C for 600 s, 45 cycles; Step 3: Melting curve at 95°C for 180 s, one cycle; followed by Step 4: Cooling at 37°C for 5 s, one cycle, then incubation. The expression levels of β-actin (internal control) were relative to the 2ΔΔCt method for analysis.

[0110] IV. Evaluation of the anti-inflammatory effect of the effective component extract of safflower compound on inflamed zebrafish

[0111] (1) Anti-inflammatory effect: The migration and other behaviors of neutrophils in live zebrafish embryos after modeling and drug administration were observed, the number of migrating neutrophils was counted, and the anti-inflammatory effect was calculated. The formula for calculating the anti-inflammatory effect of the test product is as follows:

[0112]

[0113] (2) Half-maximal effective concentration (EC50): Inflamed zebrafish embryos were immersed in solutions of 0, 125, 200, 250, 275, 500, and 1000 μg / ml of the effective fraction extract of the safflower composition for 2 h. The migration and other behaviors of neutrophils in the zebrafish embryos were observed, and the EC50 value of the effective fraction extract of the safflower composition was calculated.

[0114] (3) Zebrafish survival rate: Zebrafish embryos were immersed in HH-2 solutions of 0, 125, 250, and 500 μg / ml and cultured in a constant temperature incubator at 28.5℃. Experimental cycles were 72 h. The behavior and mortality of zebrafish in each group were observed and recorded at 0, 12, 24, 48, and 72 h. For example, failure to observe gill flapping or lack of response to touch of the caudal peduncle indicated death.

[0115] V. Statistical Analysis

[0116] GraphPad 9.0 software was used to perform one-way ANOVA for statistical analysis and data processing. Experimental data are expressed as mean ± standard error (SEM), and p < 0.05 was considered statistically significant.

[0117] VI. Effects of the effective component extracts of safflower compound on migration and aggregation in a zebrafish inflammation model

[0118] The number of neutrophils at the inflammatory site in the model group was 39, which was significantly lower than that in the control group (P < 0.0001), indicating that the copper sulfate-induced zebrafish inflammation model was successful. Furthermore, the positive control drug DEX showed an 85.73% inflammation reduction effect, indicating that the positive control drug can inhibit the migration of neutrophils to the lateral line thalamus cell region. This demonstrates that the copper sulfate-induced zebrafish inflammation model at this concentration can be curative, is highly feasible, and is suitable for drug efficacy screening.

[0119] The active ingredient extract of the safflower composition significantly inhibited neutrophil migration in a zebrafish inflammation model at a concentration of 500 μg / ml, indicating that this component has strong anti-inflammatory activity. (See...) Figure 2 .

[0120] Compared with the model group, when the effective extract concentrations of the safflower composition were 125, 250, and 500 μg / mL, the number of neutrophils in the inflamed areas of zebrafish were 35.5, 28.2, and 6.9, respectively, with inflammation reduction effects of 8.97%, 27.61%, and 82.39% (except for the 125 μg / mL concentration, all others P < 0.0001). This indicates that the anti-inflammatory effect of the HH-2 component is dose-dependent; the higher the concentration, the more significant the inhibition of neutrophil migration and the stronger the anti-inflammatory activity. See Table 2 and... Figure 3 , 4 .

[0121] VII. Dose-response effect of effective fraction extracts from safflower composition on inflamed zebrafish

[0122] Plotting neutrophil inhibition rate on the ordinate and Log10 (the effective extract of the safflower composition) on the x-axis, the EC50 of the HH-2 component was determined to be 340.5 μg / ml. At concentrations of 0, 125, 200, 250, 275, 500, and 1000 μg / mL, the number of neutrophils in the inflammatory sites of zebrafish were 39, 35.5, 34.5, 28.2, 27.7, 6.9, and 3.6, respectively. See... Figure 5 .

[0123] 8. Toxicity test of the effective component extract of the safflower composition

[0124] The survival experiment of zebrafish immersed in the test solution showed that no toxic reactions were found in zebrafish after being fed in HH-2 test solution at a dose of 0-500 μg / ml for 72 hours, which is within the safe dose range.

Claims

1. An extract of the effective fraction of a safflower composition, characterized in that, Includes the following ingredients: Syringic acid, neochlorogenic acid, deacetylated methyl ester, genipin, N-acetylated-L-tyrosine, fraxin, 4-pyridoxine, 7,8-dihydroxy-4-methylcoumarin, chlorogenic acid, ferulic acid; The total content of the above components is 80%-95% of the effective fraction extract; The aforementioned safflower composition is made from the following medicinal materials: *Hedyotis diffusa*, *Hedyotis diffusa*, *Spatholobus suberectus*, *Myrtle root*, *White-backed leaf root*, *Gardenia jasminoides*, and *Illicium verum*. Its preparation method includes the following steps: Take the safflower extract, dilute it with water by 0.8-3 times, centrifuge, take the supernatant, pass it through a C18 column, let it stand for 4-8 hours, then elute it sequentially with water (20-40 times the weight of the supernatant) and methanol (20-40 times the weight of the supernatant by volume percentage), collect the 20% methanol eluent, and vacuum dry it to obtain the final product.

2. The extract of the effective fraction of the safflower composition according to claim 1, characterized in that, The weight parts of each component in the effective fraction extract are as follows: Syringic acid 15-30 parts, neochlorogenic acid 200-300 parts, deacetylated methyl ester 8-20 parts, genipin glycoside 10-25 parts, N-acetic acid-L-tyrosine 8-20 parts, fraxin 40-60 parts, 4-pyridoxine 35-55 parts, 7,8-dihydroxy-4-methylcoumarin 180-250 parts, chlorogenic acid 200-300 parts, ferulic acid 20-40 parts.

3. The extract of the effective fraction of the safflower composition according to claim 1, characterized in that: The preparation method of the aforementioned safflower composition paste includes the following steps: The ingredients, including *Hedyotis diffusa*, ...Spatholobus suberectus*, *Rhodomyrtus tomentosa* root, *Bai Bei Ye* root, *Gardenia jasminoides*, and *Illicium verum*, are extracted with water or alcohol and then concentrated into a thick paste.

4. The extract of the effective fraction of the safflower composition according to claim 3, characterized in that: The preparation method of the aforementioned safflower composition paste includes the following steps: Take all the medicinal materials, add water equivalent to 6-12 times the weight of all the medicinal materials, and decoct 2-3 times, 1-3 hours each time. Combine the decoctions, filter, and concentrate the filtrate to a clear extract with a relative density of 1.05-1.25 at 80℃. After cooling to room temperature, add ethanol to make the alcohol content 50-80%, stir well, let stand for 12-48 hours, and filter. Recover the ethanol from the filtrate to obtain an extract with a relative density of 1.20-1.30 at 80℃.

5. The extract of the effective fraction of the safflower composition according to claim 3, characterized in that: The aforementioned safflower composition is made from the following medicinal materials in parts by weight: 400-600 parts of *Hedyotis diffusa*, 200-400 parts of *Hedyotis diffusa*, 400-600 parts of *Prunus persica*, 200-400 parts of *Bai Bei Ye Gen*, 200-400 parts of *Xi Ming*, 300-500 parts of *Ji Xue Teng*, and 400-600 parts of *Tao Jin Niang Gen*.

6. The extract of the effective fraction of the safflower composition according to claim 5, characterized in that, Its preparation method includes the following steps: Take 8%-13% of the *Illicium verum* and grind it into a fine powder for later use; add the remaining *Illicium verum* and all other medicinal materials to water equivalent to 8-12 times their weight and decoct 2-3 times, 1-3 hours each time. Combine the decoctions, filter, and concentrate to a clear extract with a relative density of 1.15-1.25 at 80℃; after cooling to room temperature, add ethanol to make the alcohol content 65%, stir well, let stand for 24 hours, and filter; recover the ethanol from the filtrate to obtain an extract with a relative density of 1.2-1.32 at 80℃, add the *Illicium verum* fine powder and mix well to obtain a mixture; Take the mixture, dilute it with water by 0.8-3 times, centrifuge, take the supernatant, pass it through a C18 column, let it stand for 4-8 hours, then elute it sequentially with water (20-40 times the weight of the supernatant) and methanol (20-40 times the weight of the supernatant by volume percentage), collect the 20% methanol eluent, and dry it under vacuum to obtain the final product.

7. Use of the extract of the effective part of the safflower composition according to any one of claims 1-6 in the preparation of an anti-inflammatory drug.

Images