Medicine with anti-inflammatory effect

Abstract

The invention provides a medicine with an anti-inflammatory effect, and relates to the field of medicines. The medicine with the anti-inflammatory effect is prepared from the following raw medicinal materials in parts by weight: 2 to 3 parts of honeysuckle flower, 0.5 to 1.5 parts of flos chrysanthemi indici, 0.5 to 1.5 parts of herba taraxaci, 0.5 to 1.5 parts of herba violae and 0.5 to 1.5 parts of radix semiaquilegiae. The preparation method comprises the following steps: mixing the raw medicinal materials with water, carrying out heating reflux extraction, carrying out solid-liquid separation, and concentrating the liquid to obtain a compound extract concentrated solution; and extracting the compound extract concentrated solution by using an organic solvent to obtain the medicine with the anti-inflammatory effect. The medicine with the anti-inflammatory effect, provided by the invention, has a remarkable anti-inflammatory effect and can be used for treating and preventing inflammations caused by various diseases.

Description

Technical Field

[0001] The present invention relates to the technical field, and more particularly to a medicine with anti-inflammatory effect. Background Art

[0002] Inflammation is a response of the body to damage caused by infection, foreign substances or other causes (such as antigen-antibody complexes). It is also the most basic pathological process of infection and many diseases, and is completed by the participation of multiple cells and cytokines. The complex process of inflammation is not only the body's resistance to external infection or damage, but also causes varying degrees of damage to the host. Many common diseases such as trauma repair, atherosclerosis, chronic obstructive pneumonia, uveitis, gastritis, enteritis, prostatitis, cystitis, etc. fall into the category of inflammation. Excessive and long-term inflammatory responses can cause damage to the body, and severe inflammation may endanger the patient's life. For example, the main physiological and pathological feature of severe cases of the new coronavirus (COVID-19) is the inflammatory storm, also known as the cytokine storm.

[0003] Inflammatory cytokines are small molecule proteins synthesized and secreted by immune cells (macrophages, T cells, B cells, etc.) and certain non-immune cells after stimulation. They play an important regulatory role in the body's immune system, mainly including Tumor Necrosis Factor-α (TNF-α), Interleukin (IL), Interferon (IFN), etc. Normal levels of TNF-α can participate in the body's resistance to bacterial, viral and parasitic infections, promote tissue repair, and cause tumor cell apoptosis, but excessive expression and release of TNF-α in the body will destroy the body's immune balance and cause a variety of diseases. Therefore, TNF-α protein is also an important target for global biopharmaceutical companies to develop anti-inflammatory drugs. In addition, interleukins are the most widely discovered type of cytokines with the most extensive regulatory effects, playing an important role in the immune system. Representative ones include IL-6, IL-1β, etc. For example, targeting IL-6 has become the main treatment strategy for immune-mediated inflammatory diseases in clinical practice. Therefore, selecting representative inflammatory cytokines such as TNF-α and IL-6 as important indicators for pharmacodynamic evaluation and developing natural drug preparations with significant anti-inflammatory effects and high safety will have broad market prospects and practical needs.

[0004] In addition, anti-inflammatory drugs are drugs used to treat inflammatory reactions that occur after tissue damage, mainly including two major categories of anti-inflammatory drugs: steroidal and non-steroidal. Steroidal anti-inflammatory drugs mainly exert their anti-inflammatory effects by inhibiting the synthesis of prostaglandins, inhibiting leukocyte aggregation, reducing bradykinin formation, and inhibiting platelet aggregation. Non-steroidal anti-inflammatory drugs are a class of anti-inflammatory drugs that do not contain steroidal structures. The commonly used ones in clinical practice mainly include salicylates (aspirin), propionic acids (ibuprofen), indoles (indomethacin), fenamic acids (flufenamic acid), acetic acids (Voltaren), etc. However, the long-term use of the above-mentioned drugs is prone to drug resistance, accompanied by gastrointestinal side effects and liver and kidney toxicity. Therefore, the development of natural medicine products with significant anti-inflammatory effects and higher safety has broad market prospects and actual needs.

[0005] Wuwei Xiaoduyin was first seen in the book "Yi Zong Jin Jian" written by Wu Qian in the Qing Dynasty (1742 AD). The whole prescription is composed of five Chinese medicinal herbs: honeysuckle, wild chrysanthemum, dandelion, Viola yedoensis, and purple back amaranth seeds. It has the effects of clearing away heat and detoxifying, and dissipating furuncles. It is widely used in multiple disciplines such as surgery, dermatology, otolaryngology, gynecology, pediatrics, and internal medicine. It is an important prescription for the treatment of furuncles and swellings. Through literature review, it was found that there is no research report on the anti-inflammatory active parts and effective monomer compounds of Wuwei Xiaoduyin compound. Summary of the invention

[0006] The purpose of this application is to provide a drug with anti-inflammatory effect to solve the above problems.

[0007] To achieve the above objectives, this application adopts the following technical solutions:

[0008] A drug with anti-inflammatory effect, characterized in that the raw medicinal materials thereof are calculated by weight and include: 2-3 parts of honeysuckle, 0.5-1.5 parts of wild chrysanthemum, 0.5-1.5 parts of dandelion, 0.5-1.5 parts of Viola yedoensis and 0.5-1.5 parts of Glechoma longituba seeds;

[0009] The preparation method thereof comprises:

[0010] The raw medicinal material is mixed with water, heated under reflux for extraction, and the liquid is concentrated after solid-liquid separation to obtain a concentrated compound extract;

[0011] The compound extract concentrate is extracted using an organic solvent to obtain the medicine with anti-inflammatory effect.

[0012] Optionally, among the raw medicinal materials, calculated by weight, honeysuckle can be 2 parts, 2.5 parts, 3 parts or any value between 2-3 parts; wild chrysanthemum can be 0.5 parts, 1 parts, 1.5 parts or any value between 0.5-1.5 parts; dandelion can be 0.5 parts, 1 parts, 1.5 parts or any value between 0.5-1.5 parts; Viola yedoensis can be 0.5 parts, 1 parts, 1.5 parts or any value between 0.5-1.5 parts; and Gynura ovata can be 0.5 parts, 1 parts, 1.5 parts or any value between 0.5-1.5 parts.

[0013] Preferably, the amount of water used is 9-11 times the mass of the raw medicinal material.

[0014] Optionally, the amount of water used can be 9 times, 10 times, 11 times or any value between 9 and 11 times the mass of the raw medicinal material.

[0015] Preferably, the organic solvent includes one or more of petroleum ether, ethyl acetate, and n-butanol.

[0016] Preferably, the organic solvent is petroleum ether and / or ethyl acetate.

[0017] Preferably, the drug comprises fat-soluble components and medium-polar caffeoyl components.

[0018] Preferably, the medium polar caffeoyl component includes isochlorogenic acid C.

[0019] Preferably, the drug with anti-inflammatory efficacy further comprises an auxiliary material.

[0020] Preferably, the excipients include one or more of stearic acid, glyceryl monostearate, sodium lauryl sulfate, aluminum distearate, sorbitan sesquioleate, polyethylene glycol, propylene glycol, sorbitol, liquid paraffin, vaseline, lanolin, beeswax, vegetable oil, rosin, zinc oxide, sodium carboxymethyl cellulose, sodium alginate, tragacanth gum, and gelatin.

[0021] Preferably, the drug with anti-inflammatory efficacy has a dosage form including any one of ointment, gel, patch, paste, and liniment.

[0022] Preferably, the inflammatory factors targeted by the drug include tumor necrosis factor-α and interleukin-6.

[0023] Compared with the prior art, the beneficial effects of this application include:

[0024] The drug with anti-inflammatory effect provided in the present application takes the classic compound of traditional Chinese medicine Wuwei Xiaoduyin as the research object, and further obtains the components containing compounds of different polarities by using the water extraction and concentration method to obtain the compound decoction, and screens the cytotoxicity and anti-inflammatory activity in vitro. For the first time, it was determined that the fat-soluble components and the medium-polarity caffeoyl components in the compound are the key active components of the compound to exert anti-inflammatory effects, which can significantly inhibit the expression of TNF-α and IL-6 in macrophages induced by lipopolysaccharide (LPS), and exert anti-inflammatory activity. Furthermore, the main monomer compounds in the medium polarity were quickly obtained by column chromatography combined with high-performance liquid chromatography (HPLC), and the structure was identified by mass spectrometry, nuclear magnetic resonance and other spectral techniques. And the monomer compound isochlorogenic acid C was determined for the first time by the in vitro activity screening method as the key small molecule compound for the active component to exert anti-inflammatory effects, and the compound can be used as an important quality control indicator for anti-inflammatory related products.

[0025] This application provides a solvent extraction method for the first time, which can quickly and efficiently obtain two types of key anti-inflammatory active components, fat-soluble and medium-polarity, from the water extract of the Wuwei Xiaoduyin compound. The medium-polarity anti-inflammatory active components are mainly caffeoyl chemical components, especially the natural product isochlorogenic acid C, which is the key small molecule compound for the anti-inflammatory activity of this component and can be used as a key indicator for quality control. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope of the present application.

[0027] Figure 1 (a) shows the HPLC analysis results of sample WWXD-H2O;

[0028] Figure 1(b) is the HPLC analysis result of sample JYH-H2O;

[0029] Figure 1(c) is the HPLC analysis result of sample YJH-H2O;

[0030] Figure 1(d) shows the HPLC analysis results of sample ZHDD-H2O;

[0031] Figure 1(e) is the HPLC analysis result of sample PGY-H2O;

[0032] Figure 1(f) is the HPLC analysis result of sample TKZ-H2O;

[0033] Figure 2(a) is the UV absorption characteristic diagram of WWXD-1 and WWXD-2;

[0034] Figure 2(b) is the UV absorption characteristic diagram of WWXD-3 and WWXD-4;

[0035] Figure 2(c) is the UV absorption characteristic diagram of WWXD-5 and WWXD-6;

[0036] Figure 2(d) is the UV absorption characteristic diagram of WWXD-7 and WWXD-8;

[0037] Figure 2(e) is the UV absorption characteristic diagram of WWXD-9, WWXD-10 and WWXD-11;

[0038] Figure 3 (a) is the HPLC analysis result of WWXD-PE;

[0039] Figure 3(b) is the HPLC analysis result of WWXD-EA;

[0040] FIG3( c ) is the HPLC analysis result of WWXD-BU;

[0041] Figure 3(d) is the HPLC analysis result of WWXD-C-H2O;

[0042] Figure 4 The results of cytotoxicity tests of the aqueous extract of the compound and different extraction parts are shown;

[0043] Figure 5 The results of the anti-inflammatory activity test of the water extract of the compound and different extraction parts are shown;

[0044] Figure 6 These are the test results of the inhibitory activity of the characteristic monomer compounds WWXD-4, 6, 9, and 10 on the increase of TNF-α in RAW264.7 cells induced by LPS. DETAILED DESCRIPTION

[0045] The embodiments of the present application will be described in detail below in conjunction with specific examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present application and should not be considered as limiting the scope of the present application. If specific conditions are not specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0046] First, the materials, reagents and instruments used in the examples of the present application are described, as shown in Table 1, Table 2 and Table 3 below:

[0047] Table 1 Sources of Chinese herbal medicine pieces

[0048] Name of medicinal material factory Origin batch number honeysuckle Guangzhou Zhixin Chinese Medicine Pieces Co., Ltd. Henan 210801 Wild Chrysanthemum Guangzhou Zhixin Chinese Medicine Pieces Co., Ltd. Anhui 220101 Dandelion Yulin Bencaotang Chinese Medicine Pieces Co., Ltd. Anhui 220602 Viola yedoensis Zhixin (Zunyi) Chinese Medicine Pieces Co., Ltd. Jiangsu 220501 Glechoma longituba Guangzhou Zhixin Chinese Medicine Pieces Co., Ltd. Jiangsu 201201

[0049] Table 2 Reagents

[0050]

[0051]

[0052] Table 3 Main equipment and consumables

[0053] Device Name Manufacturer Low temperature freeze dryer Christ Company, Germany Rotary evaporator BUCHI AG Electric heating jacket Shanghai Heqi Glass Instrument Co., Ltd. Agilent 1260 HPLC Agilent Corporation Multifunctional ELISA reader Swiss Tecan Electronic precision balance Mettler Toledo, Switzerland PCR Thermal Cycler Thermo Fisher Agilent G6520Q-TOF Mass Spectrometer Agilent Technologies Agilent ZORBAX Columns Agilent Corporation Bruker AvanceⅢ500 NMR Bruker AG

[0054] Example 1

[0055] This embodiment provides a drug with anti-inflammatory effect, and its preparation method is as follows:

[0056] Weigh the following Chinese herbal medicine pieces respectively: 10 g of honeysuckle, 4 g of wild chrysanthemum, 4 g of dandelion, 4 g of viola yedoensis, and 4 g of gynura strychnifolia. Place the above Chinese herbal medicine pieces in a 1L round-bottom flask, place the round-bottom flask in a heating mantle, add 10 times the amount of distilled water in the amount of the total mass of the pieces, soak for 30 minutes, then heat and reflux for extraction. After the water temperature reaches 100 degrees, continue heating for 1 hour. After the heating is completed, filter while hot, collect the filtrate, add 10 times the amount of distilled water to the residue again, repeat the above reflux extraction, combine the two filtrates, and concentrate under reduced pressure to obtain 100 ml of compound concentrate.

[0057] The extract concentrate was placed in a 1L separatory funnel, and equal volumes of petroleum ether, ethyl acetate, and n-butanol solvents were added in sequence for extraction. The extraction was repeated 5 times with each solvent, and the upper extracts were combined and concentrated under reduced pressure to obtain petroleum ether, ethyl acetate, and n-butanol extract concentrates in sequence. The weights after freeze-drying were 12.5 mg of petroleum ether extract (WWXD-PE), 845.4 mg of ethyl acetate extract (WWXD-EA), 1438.8 mg of n-butanol extract (WWXD-BU), and 4667.5 mg of the aqueous layer sample after extraction (WWXD-C-H2O).

[0058] In order to detect the compound concentrate, the sample preparation of each single herbal extract and the construction of HPLC fingerprint, the following experiments were carried out:

[0059] Weigh the following Chinese herbal medicine pieces respectively: 10 g of honeysuckle, 4 g of wild chrysanthemum, 4 g of dandelion, 4 g of Viola yedoensis, and 4 g of Gynura nigra. Place the above Chinese herbal medicine pieces in a 1L round-bottom flask, place the round-bottom flask in a heating mantle, add distilled water 10 times the total mass of the pieces, soak for 30 minutes, then heat and reflux for extraction. After the water temperature reaches 100 degrees, continue heating for 1 hour. After the heating is completed, filter while hot, collect the filtrate, add 10 times the amount of distilled water to the residue again, repeat the above reflux extraction, combine the two filtrates, and concentrate under reduced pressure to obtain 50 ml of compound concentrate, named WWXD-H2O.

[0060] Weigh 30 g of the Chinese herbal medicine piece honeysuckle and place it in a 1L round-bottom flask, add distilled water 10 times the total mass of the piece, soak for 30 minutes, then heat and reflux extraction, and continue heating for 1 hour after the water temperature reaches 100 degrees. After the heating extraction is completed, filter while hot and collect the filtrate. Repeat the above reflux extraction with the residue, combine the two filtrates, and concentrate under reduced pressure to obtain 50 ml of honeysuckle water extract concentrate, named JYH-H2O.

[0061] Weigh 30 g of wild chrysanthemum in a 1L round-bottom flask, add 10 times the total mass of distilled water, soak for 30 min, then heat and reflux for extraction. After the water temperature reaches 100 degrees, continue heating for 1 h. After the heating extraction is completed, filter while hot and collect the filtrate. Repeat the reflux extraction with the residue, combine the two filtrates, and concentrate under reduced pressure to obtain 50 ml of wild chrysanthemum water extract concentrate, named YJH-H2O.

[0062] Weigh 30 g of the Chinese herbal medicine dandelion and place it in a 1L round-bottom flask, add distilled water 10 times the total mass of the herbal medicine, soak for 30 minutes, then heat and reflux for extraction. After the water temperature reaches 100 degrees, continue heating for 1 hour. After the heating extraction is completed, filter while hot and collect the filtrate. Repeat the above reflux extraction with the residue, combine the two filtrates, and concentrate under reduced pressure to obtain 50 ml of dandelion water extract concentrate, named PGY-H2O.

[0063] Weigh 30 g of the Chinese herbal medicine Viola yedoensis and place it in a 1L round-bottom flask, add distilled water 10 times the total mass of the herbal medicine, soak for 30 minutes, then heat and reflux for extraction. After the water temperature reaches 100 degrees, continue heating for 1 hour. After the heating extraction is completed, filter while hot and collect the filtrate. Repeat the above reflux extraction with the residue, combine the two filtrates, and concentrate under reduced pressure to obtain 50 ml of Viola yedoensis aqueous extract concentrate, named ZHDD-H2O.

[0064] Weigh 30 g of the Chinese herbal medicine piece Gynura odoratum seeds and place it in a 1L round-bottom flask, add distilled water 10 times the total mass of the piece, soak for 30 minutes, then heat and reflux for extraction. After the water temperature reaches 100 degrees, continue heating for 1 hour. After the heating extraction is completed, filter while hot and collect the filtrate. Repeat the above reflux extraction with the residue, combine the two filtrates, and concentrate under reduced pressure to obtain 50 ml of Gynura odoratum seeds water extract concentrate, named TKZ-H2O.

[0065] Accurately pipette 1 ml of each of the concentrated samples WWXD-H2O, JYH-H2O, YJH-H2O, PGY-H2O, ZHDD-H2O, and TKZ-H2O in sequence, place them in a 10 ml volumetric flask, add distilled water to make up to 10 ml, and take an appropriate amount of solution after it is completely dissolved and filter it with a microporous filter membrane for later use. Using the following HPLC chromatographic conditions, the 11 characteristic chemical components in the compound Wuwei Xiaodu Yin and each single Chinese medicine water extract can be quickly identified. The specific situation is as follows Figure 1(a)-Figure 1(f)As shown, WWXD-H2O, namely FF-H2O, is shown in Figure 1(a), JYH-H2O is shown in Figure 1(b), YJH-H2O is shown in Figure 1(c), ZHDD-H2O is shown in Figure 1(d), PGY-H2O is shown in Figure 1(e), and TKZ-H2O is shown in Figure 1(f). The HPLC analysis method has the advantages of high recognition, simple operation, and strong pertinence for the characteristic chemical components in WWXD-H2O. The analysis method and conditions (hereinafter referred to as condition 1) are as follows: Agilent 1260 liquid chromatography instrument, C18 (5μm, 4.6×250mm, Agilent: ZORBAX SB) chromatographic column, column temperature 25℃, flow rate 1.0mL / min; detection wavelength 190~400nm full wavelength scan; injection volume 10μL. The mobile phase is methanol-water (containing 0.2% acetic acid). The specific chromatographic conditions are shown in Table 4:

[0066] Table 4 HPLC analysis method of compound and single herbal medicine water extract

[0067]

[0068] The detection wavelength of 320 nm, where the characteristic chemical components in WWXD-H2O have strong ultraviolet absorption, was selected to analyze the characteristic chemical components in the compound and single herbal water extracts. The results showed that the compound WWXD-H2O mainly contained 11 characteristic chemical components, which were numbered WWXD-1 to 11 in sequence according to the retention time. Among them, there were 6 common chemical components with high content (WWXD-2, WWXD-4, WWXD-5, WWXD-8, WWXD-9 and WWXD-10) in wild chrysanthemum (YJH-H2O) and honeysuckle (JYH-H2O), and 1 characteristic compound (WWXD-11) in YJH-H2O; Viola yedoensis (ZHDD-H2O) contributed two characteristic compounds (WWXD-3 and WWXD-6); PGY-H2O contributed 1 characteristic compound (WWXD-1) and 1 monomer compound (WWXD-4) shared by YJH-H2O and JYH-H2O. The specific chromatographic analysis results are shown in Figure 1(a)-Figure 1(f) The UV absorption characteristic diagrams of the 11 characteristic chemical components show that compounds WWXD-1, WWXD-2, WWXD-4, WWXD-5, WWXD-7 to 10 are all caffeic acid compounds, and compound WWXD-11 is a flavonoid compound. The specific UV absorption characteristic diagrams of the 11 characteristic chemical components are shown in Figure 2(a)-Figure 2(e), WWXD-1 and WWXD-2 are shown in Figure 2(a), WWXD-3 and WWXD-4 are shown in Figure 2(b), WWXD-5 and WWXD-6 are shown in Figure 2(c), WWXD-7 and WWXD-8 are shown in Figure 2(d), and WWXD-9, WWXD-10, and WWXD-11 are shown in Figure 2(e).

[0069] Weigh 1 mg of each of the four samples of WWXD-PE, WWXD-EA, WWXD-BU, and WWXD-C-H2O accurately, dissolve and dilute in a 2 ml volumetric flask for later use. Using the same HPLC chromatographic conditions as above (condition 1), analyze the correlation between the main components in the four samples and the main components in the compound water extract (WWXD-H2O). The distribution of the 11 characteristic chemical components in WWXD-H2O in the four samples was analyzed and compared by the retention time of the characteristic compounds and the UV characteristic diagram. The specific analysis results are as follows: Figure 3(a)-Figure 3(d) As shown in Figure 3(a), WWXD-PE is shown in Figure 3(b), WWXD-EA is shown in Figure 3(c), and WWXD-C-H2O is shown in Figure 3(d). By comparison, it was found that the 11 characteristic compounds in WWXD-H2O were mainly distributed in the two extraction parts of WWXD-EA and WWXD-BU, among which the characteristic compounds in the WWXD-EA part were the most abundant, totaling 7. The two samples WWXD-PE and WWXD-C-H2O were mainly composed of volatile components without UV absorption and sugar chemical components, respectively.

[0070] The components of the compound extract and each extraction part were evaluated as follows:

[0071] 1. Safety and anti-inflammatory activity screening of compound extracts and each extraction part

[0072] (1) CCK-8 method to test the cytotoxicity of the compound and the extracted parts

[0073] RAW264.7 cells were cultured in an incubator at 37°C and 5% CO2. The cells were passaged when they grew to 70% to 80%. RAW264.7 cells in the logarithmic growth phase were inoculated in a 96-well culture plate at 1×105 / mL, with 100 μL per well. The cytotoxicity of WWXD-H2O, WWXD-PE, WWXD-EA, WWXD-BU and WWXD-C-H2O at mass concentrations of 0, 1.56, 3.12, 6.25, 12.5, 25, 50, 100, and 200 μg / mL was measured respectively. Three replicate wells were set for each sample. After culturing for 24 hours at 37°C and 5% CO2, 10 μL of CCK-8 solution was added to each well. After culturing for 1 hour, the absorbance A at 450 nm was measured using an enzyme reader, and repeated three times. The cell survival rate was calculated according to the formula: cell survival rate = (A treatment group - A negative group) / (A blank group - A negative group) × 100%. The test results showed that WWXD-H2O, WWXD-BU and WWXD-C-H2O showed no cytotoxicity at the set maximum concentration of 200μg / mL, and the safe doses of WWXD-PE and WWXD-EA samples were 0-12.5μg / mL and 0-50μg / mL, respectively. For specific test results, see Figure 4 .

[0074] (2) Screening of anti-inflammatory activity of compound extracts and their extraction parts

[0075] The anti-inflammatory activity test was divided into 8 groups, namely, blank group, model group, positive drug group (dexamethasone-DEX, added at a concentration of 5 μg / mL), WWXD-H2O group (added at a concentration of 200 μg / mL), WWXD-PE group (added at a concentration of 12.5 μg / mL), WWXD-EA group (added at a concentration of 50 μg / mL), WWXD-BU group (added at a concentration of 200 μg / mL) and WWXD-C-H2O group (added at a concentration of 200 μg / mL). The anti-inflammatory initial screening concentration of each drug group selected the highest safe dose of CCK-8 cytotoxicity test. RAW264.7 cells in the logarithmic growth phase were taken and 1×10 5The cells were seeded at a density of 1 μg / mL in a 96-well plate and placed in an incubator for 24 hours before administration. Complete culture medium was added to the blank group, 1 μg / mL of LPS was added to the model group, the positive drug group and each drug-administered group, 5 μg / mL of dexamethasone was given to the positive drug group, and the drug-administered groups were given the highest concentration of drugs at each safe dose according to the results of the cytotoxicity test, and acted together for 24 hours. The ELISA method was used to detect the effect of the drug-administered group on the release of TNF-α in the supernatant of RAW264.7 cells induced by LPS. The test results showed that WWXD-PE (12.5 μg / mL), WWXD-EA (50 μg / mL), WWXD-BU (200 μg / mL) and WWXD-C-H2O (200 μg / mL) could significantly inhibit the expression of TNF-α in the supernatant of RAW264.7 cells induced by LPS at a safe dose, and had anti-inflammatory activity (test results see Figure 5 Among them, WWXD-PE and WWXD-EA have stronger inhibitory activity and lower effective concentration than other components.

[0076] On the basis of the above experiments, WWXD-PE and WWXD-EA samples with significant anti-inflammatory activity were further selected for anti-inflammatory concentration-dependent tests. The WWXD-PE group was set up with two dosing groups of high dose (12.5μg / mL) and low dose (5μg / mL), and the WWXD-EA group was set up with three concentration doses of high (45μg / mL), medium (15μg / mL), and low (5μg / mL). The specific experimental operations of the blank group, model group, positive drug group (DEX, 5μg / mL) and each dosing group were consistent with the above anti-inflammatory test. The ELISA method was also used to detect the effects of the dosing groups on the release of TNF-α and IL6 in the supernatant of RAW264.7 cells induced by LPS. The test results showed that both high-dose (12.5 μg / mL) and low-dose (5 μg / mL) WWXD-PE could significantly inhibit the expression of TNF-α in the supernatant of RAW264.7 cells induced by LPS, and its activity was equivalent to that of positive drugs; high-dose (12.5 μg / mL) WWXD-PE could significantly inhibit the increase of IL6 (test results see Figure 5 (B), (D)). High (45μg / mL) and medium (15μg / mL) doses of WWXD-EA could significantly inhibit the expression of TNF-α in the supernatant of RAW264.7 cells induced by LPS, while low dose (5μg / mL) had no significant inhibitory activity; high (45μg / mL) and medium (15μg / mL) doses of WWXD-EA could significantly inhibit the increase of IL6 (test results see Figure 5 (C), (E)).

[0077] In summary, the active substances in the Wuwei Xiaoduyin compound that exert anti-inflammatory effects are mainly present in the fat-soluble (WWXD-PE) and medium-polar (WWXD-EA) parts. The WWXD-EA part is safer than WWXD-PE.

[0078] It should be noted that Figure 5 In the figure, (A) is the test results of the inhibitory activity of the compound water extract and different extraction parts at the highest safe dose on the increase of TNF-α in RAW264.7 cells induced by LPS; (B) is the inhibitory activity of WWXD-PE at high (12.5μg / mL) and low (5μg / mL) concentrations on the increase of TNF-α; (C) is the inhibitory activity of WWXD-EA at high (45μg / mL), medium (15μg / mL) and low (5μg / mL) concentrations on the increase of TNF-α; (D) is the inhibitory activity of WWXD-PE at high (12.5μg / mL) and low (5μg / mL) concentrations on the increase of inflammatory factor IL6; (E) is the inhibitory activity of WWXD-EA at high (45μg / mL), medium (15μg / mL) and low (5μg / mL) concentrations on the increase of IL6.

[0079] 2. Isolation, purification and activity testing of the main characteristic chemical components in the compound water extract

[0080] According to the above anti-inflammatory activity test results, combined with the chemical composition analysis data of different extraction parts of the compound, the monomer compounds with high safety and good anti-inflammatory activity in the compound are mainly present in the WWXD-EA part. Comparison of WWXD-EA and WWXD-BU Monomer compounds WWXD-6, 9, and 10 are components with higher content in WWXD-EA, and WWXD-4 is the monomer compound with the highest content in WWXD-BU. Since WWXD-2 and 3 are mainly present in WWXD-BU, and the contents of WWXD-8 and 11 in WWXD-EA and WWXD-BU are similar, and the anti-inflammatory activity of WWXD-BU is significantly weaker than that of WWXD-EA, the next step will be to prioritize the separation and purification of the four monomer compounds WWXD-4, 6, 9, and 10 and evaluate their anti-inflammatory activity.

[0081] Weigh 100g of Chinese medicine slices of dandelion and place them in a 10L round-bottom flask, add 10 times the total mass of the slices of distilled water, soak for 30min, then heat and reflux for extraction, continue heating for 1h after the water temperature reaches 100 degrees, filter while hot after the extraction is completed, collect the filtrate, add 10 times the amount of distilled water to the residue again, repeat the above reflux extraction, combine the filtrates twice, and concentrate under reduced pressure to obtain 300ml of dandelion water extract concentrate for standby use. The extracted concentrate was placed in a 1L separatory funnel, and equal volumes of petroleum ether, ethyl acetate, and n-butanol solvents were added in sequence for extraction. Each solvent was extracted 5 times, and the upper extract was combined and concentrated under reduced pressure to obtain petroleum ether (PGY-PE), ethyl acetate (PGY-EA), and n-butanol (PGY-BU) extract concentrates in sequence. The above samples and the water layer samples after extraction were analyzed by HPLC, and the chromatographic method was consistent with the aforementioned conditions (condition 1). The analysis results showed that the characteristic compounds WWXD-1 and WWXD-4 in the compound were both present in the water layer after extraction. Therefore, the water layer sample of the dandelion water extract was extracted and concentrated to 200 ml under reduced pressure, and then eluted with D101 macroporous resin and ethanol-water gradient to obtain five components: water, 20% ethanol, 40% ethanol, 60% ethanol, and 95% ethanol. HPLC analysis showed that WWXD-1 and WWXD-4 were mainly present in the water elution component. The D101 water elution component sample was repeatedly purified by ODS filler methanol-water to finally obtain pure WWXD-4 (31 mg). The quasi-molecular ion peak m / z of WWXD-4 was 353 ([MH] - ), indicating a molecular weight of 354. The results of HPLC ultraviolet full wavelength scanning (DAD) showed that the compound had typical ultraviolet absorption characteristics of caffeoyl, and WWXD-4 was confirmed to be chlorogenic acid by comparison with chlorogenic acid standards using HPLC.

[0082] Weigh 40g of Chinese medicine slices of Viola yedoensis and place them in a 1L round-bottom flask, add 10 times the total mass of the slices of distilled water, soak for 30min, and then heat and reflux at 100 degrees for extraction. After the extraction is completed, filter while hot, collect the filtrate, add 10 times the amount of distilled water to the residue again, repeat the above method, combine the 2 extracts, and concentrate under reduced pressure to obtain 100ml of Viola yedoensis water extract concentrate. Use equal volumes of petroleum ether, ethyl acetate, and n-butanol solvents for extraction in turn, and each solvent is repeatedly extracted 5 times. Combine the upper extracts and concentrate under reduced pressure to obtain petroleum ether (ZHDD-PE), ethyl acetate (ZHDD-EA), and n-butanol (ZHDD-BU) extract concentrates in turn. The above samples were analyzed by HPLC, and compounds WWXD-3 and WWXD-6 were mainly present in the ZHDD-BU component. ZHDD-BU (2969mg) component was analyzed by HPLC after D101 ethanol-water gradient elution, and WWXD-3 and WWXD-6 were respectively present in the 40% and 60% ethanol elution components. The above components were repeatedly purified by ODS filler methanol-water to finally obtain pure WWXD-6 (74.3 mg). The molecular weight of WWXD-6 was confirmed by ESI-MS mass spectrometry to be 178 (quasi-molecular ion peak m / z [MH] - :177). 1H-NMR (DMSO, 500MHz) test results δH (ppm): 7.84 (1H, d, J = 9.43Hz), 6.96 (1H, s), 6.73 (1H, s), 6.15 (1H, d, J = 9.43Hz); 13C-NMR (DMSO, 125MHz) test results δC (ppm): 161.4 (C-2), 150.9 (C-7), 148.9 (C-9), 145.0 (C-4), 143.4 (C-6), 112.7 (C-5), 111.9 (C-3), 111.2 (C-10), 103.0 (C-8). The NMR test results of WWXD-6 were basically consistent with those reported in the literature (Zhang Dongbo et al., Chinese Herbal Medicine, 2019, 50(12): 2798-2801), and WWXD-6 was identified as quinoline.

[0083] Weigh 40g of Chinese medicine slices of wild chrysanthemum and place them in a 1L round-bottom flask, add 10 times the total mass of the slices of distilled water, soak for 30min, heat and reflux at 100 degrees for extraction, filter while hot after extraction, collect the filtrate, add 10 times the amount of distilled water to the residue again, repeat the above method, combine the 2 extracts, and concentrate under reduced pressure to obtain 100ml of wild chrysanthemum water extract concentrate. Similarly, solvent extraction method is used to obtain petroleum ether (YJH-PE), ethyl acetate (YJH-EA), and n-butanol (YJH-BU) extract concentrates in turn. The above samples were analyzed by HPLC, and compounds WWXD-7~11 were mainly present in the YJH-BU component. YJH-BU (875mg) was analyzed by HPLC after D101 ethanol-water gradient elution, WWXD-7~10 was mainly present in the 40% ethanol elution component, and WWXD-11 was mainly present in the 60% ethanol elution component. The 40% ethanol elution fraction was repeatedly purified by ODS filler methanol-water, and finally obtained two monomer compounds WWXD-9 (27.8 mg) and WWXD-10 (17.9 mg). The quasi-molecular ion peak m / z of WWXD-9 and WWXD-10 was 515 ([MH] - ), indicating that the molecular weight of both compounds is 516. 1H-NMR (CD3OD, 500MHz) test results δH (ppm): 8.28 (1H, d, J = 15.8Hz), 8.23 ​​(1H, d, J = 15.8Hz), 7.83 (1H, d, J = 1.7Hz), 7.82 (1H, d, J = 1.1Hz), 7.76 (2H, m), 7.55 (2H, dd, J = 1.7, 8.2Hz), 7.05 (1H, d, J = 15.8Hz), 6.96 (1H, d, J = 15.8Hz), 5.76 (1H, m), 4.98 (1H, m), 3.97 (1H, m), 1.50-3.00 (quinic acidmoiety, 4H, m); 13C-NMR (CD3OD, 125MHz) test results δC (ppm): 176.2 (C-7), 167.3 (C-1'), 166.9 (C-1"), 149.7 (C-6', 6"), 146.8 (C-7', 7", 3', 3"), 126.7 (C-4', 4"), 122.8 (C -9',9"), 117.0(C-8',8"), 115.9(C-5',5"), 115.1(C-2'), 114.9(C-2"), 75.2(C-1,4), 70.8(C-3), 69.0(C-5), 40.6(C-6, overlapped), 38.7(C-2). WWXD-10 NMR data and literature (Jun Li, et al. Natural Product Research, 2009, 23(15): 1378-1383) reported that WWXD-10 was identified as isochlorogenic acid C. According to the ultraviolet absorption characteristics and mass spectrometry data of WWXD-9 and WWXD-10, combined with the HPLC peak retention time and reference (Shen Han et al., Chinese Patent Medicine, 2021, 43(1): 22-28), WWXD-9 can be determined to be isochlorogenic acid A.

[0084] The anti-inflammatory activity test of the monomer compound was consistent with the anti-inflammatory activity test of the compound extract and each extraction part. The number of groups was set to 7 groups, namely blank group, model group, positive drug group (DEX, 5μg / mL), WWXD-4 (100μM), WWXD-6 (100μM), WWXD-9 (100μM) and WWXD-10 (100μM) monomer compound group. The ELISA method was used to detect the effect of the drug administration group on the release of TNF-α in the supernatant of RAW264.7 cells induced by LPS. The test results showed that WWXD-10 at 100μM could significantly inhibit the expression of TNF-α in the supernatant of RAW264.7 cells induced by LPS (**P<0.01), and had anti-inflammatory activity; WWXD-4 and 6 at 100μM could significantly inhibit the expression of TNF-α in the supernatant of RAW264.7 cells induced by LPS (*P<0.05), and had anti-inflammatory activity. In general, WWXD-10 has stronger anti-inflammatory activity. Therefore, the three chemical components of WWXD-4, 6, and 10 can be used as the key anti-inflammatory active ingredients in the compound five-flavor disinfection drink for quality control to ensure product quality. Figure 6 shown.

[0085] Example 2

[0086] According to the content of dexamethasone in dexamethasone acetate cream (0.75 mg / g), combined with the anti-inflammatory activity test results of the WWXD-EA extraction site, the content of WWXD-EA in the ointment was set to 7.5 mg / g. The specific ointment preparation process is as follows:

[0087] Weigh 5 g of stearic acid, 7.5 g of liquid paraffin, and 1 g of lanolin, put them in a 100 ml beaker, add 7.5 mg of WWXD-EA30, heat in a water bath and stir thoroughly to melt to obtain an oil phase; weigh 2.5 g of glycerol, 10 g of distilled water and 15 g of triethanolamine, put them in a 100 ml beaker, heat and fully dissolve to obtain an aqueous phase, and an appropriate amount of methyl paraben can be added to the aqueous phase for preservation; mix the aqueous phase and the oil phase thoroughly while hot, stir continuously until cooled to room temperature, fill and seal the tube, and an external ointment preparation with the compound anti-inflammatory active part as raw material can be obtained.

[0088] The present application provides a drug with anti-inflammatory effect, which is a compound composed of five Chinese medicines, namely honeysuckle (JYH), wild chrysanthemum (YJH), dandelion (PGY), Viola yedoensis (ZHDD), and purple back sky amaranth (ZBTK), extracted by heating and refluxing at 100°C in water as an extraction solvent, and the filtrate is filtered and concentrated under reduced pressure to obtain a compound extract concentrate. Further, the anti-inflammatory active components can be quickly and efficiently obtained from the compound extract concentrate by solvent extraction. Furthermore, the anti-inflammatory active small molecule compound isochlorogenic acid C can be obtained from the anti-inflammatory active components by column chromatography combined with HPLC analysis technology. The compound is a classic prescription for the treatment of furunculosis and swelling in traditional Chinese medicine, but so far, there has been no research on the anti-inflammatory active parts and effective monomer compounds of the Wuwei Xiaoduyin compound.

[0089] The inventors of this application provide a solvent extraction method for the first time, which can quickly and efficiently obtain two types of key anti-inflammatory active components, fat-soluble and medium-polarity, from the water extract of the Wuwei Xiaoduyin compound. The medium-polarity anti-inflammatory active components are mainly caffeoyl chemical components, especially the natural product isochlorogenic acid C, which is the key small molecule compound for the anti-inflammatory activity of this component and can be used as a key indicator for quality control.

[0090] Although some specific embodiments of the present invention have been described in detail by way of example, it will be appreciated by those skilled in the art that the above examples are for illustration only and are not intended to limit the scope of the present invention. It will be appreciated by those skilled in the art that the above embodiments may be modified without departing from the scope and spirit of the present invention. The scope of the present invention is defined by the appended claims.

Claims

1. A drug with anti-inflammatory effect, characterized in that: The raw medicinal materials are calculated by weight and include: 2-3 parts of honeysuckle, 0.5-1.5 parts of wild chrysanthemum, 0.5-1.5 parts of dandelion, 0.5-1.5 parts of Viola yedoensis and 0.5-1.5 parts of Glechoma longituba seeds; The preparation method thereof comprises: The raw medicinal material is mixed with water, heated under reflux for extraction, and the liquid is concentrated after solid-liquid separation to obtain a concentrated compound extract; The compound extract concentrate is extracted using an organic solvent to obtain the medicine with anti-inflammatory effect.

2. The drug with anti-inflammatory effect according to claim 1, characterized in that The amount of water used is 9-11 times the mass of the raw medicinal material.

3. The drug with anti-inflammatory effect according to claim 1, characterized in that The organic solvent includes one or more of petroleum ether, ethyl acetate, and n-butanol.

4. The drug with anti-inflammatory effect according to claim 3, characterized in that The organic solvent is petroleum ether and / or ethyl acetate.

5. The drug with anti-inflammatory effect according to claim 1, characterized in that The final product comprises fat-soluble components and medium-polar caffeoyl components.

6. The drug with anti-inflammatory effect according to claim 5, characterized in that The medium polar caffeoyl components include isochlorogenic acid C.

7. The drug with anti-inflammatory effect according to claim 1, characterized in that Also includes auxiliary materials.

8. The drug with anti-inflammatory effect according to claim 7, characterized in that: The auxiliary materials include one or more of stearic acid, glyceryl monostearate, sodium lauryl sulfate, aluminum distearate, sorbitan sesquioleate, polyethylene glycol, propylene glycol, sorbitol, liquid paraffin, vaseline, lanolin, beeswax, vegetable oil, rosin, zinc oxide, sodium carboxymethyl cellulose, sodium alginate, tragacanth gum, and gelatin.

9. The drug with anti-inflammatory effect according to claim 1, characterized in that The dosage form includes any one of ointment, gel, patch, paste and liniment.

10. The drug with anti-inflammatory effect according to any one of claims 1 to 9, characterized in that: The drug targets inflammatory factors including tumor necrosis factor-α and interleukin-6.

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