Preparation method and application of honeysuckle extract

Abstract

The present application relates to a kind of preparation method and application of Lonicera japonica extract, the preparation method is carried out according to the following steps: Lonicera japonica crude drug is decocted with water, filtered, concentrated;Organic solvent extraction, retain organic layer, the organic layer is concentrated to dry, hot water extraction, suction filtration, retain water layer;Water layer acidification ether extraction, retain ether layer;Ether layer is extracted with sodium bicarbonate solution, retain sodium bicarbonate extract;Sodium bicarbonate extract is acidified, concentrated, crystallized, CHCl3 recrystallization, obtain Lonicera japonica extract, i.e. 3,4-dihydroxybenzoic acid.The method of the present application fully extracts 3,4-dihydroxybenzoic acid in Lonicera japonica, improves the utilization rate of Lonicera japonica, saves medicinal materials;The Lonicera japonica extract has the effects of anti-inflammatory and antibacterial, can synergistically enhance when combined with antibiotics, reduce the amount of antibiotics, prevent the occurrence of bacterial diseases of livestock and poultry;The Lonicera japonica extract can be used as feed additive, suitable for various livestock and poultry.

Description

Technical Field

[0001] This invention belongs to the field of natural product chemistry technology, specifically relating to a method for preparing and applying honeysuckle vine extract. Background Technology

[0002] Protocatechuic acid, with the molecular formula 3,4-dihydroxybenzoic acid and a molecular weight of 154.12, is a natural phenolic acid widely found in medicinal herbs, vegetables, and fruits. It is a white to brown crystalline powder (discoloring in air), soluble in ethanol, ether, and hot water, decomposes in boiling water releasing carbon dioxide, is slightly soluble in cold water (1:50), and insoluble in benzene. Its relative density is 1.54 g / cm³. 3 It has a melting point of approximately 200℃ (decomposes). As one of the main metabolites of anthocyanins in the body, protocatechuic acid has a wide range of pharmacological activities, including antioxidant, antibacterial, anti-inflammatory, and antiviral effects. It can be used as an antioxidant, anti-inflammatory drug, neuroprotective agent, antitumor drug, antibacterial drug, antidiabetic drug, and anti-apoptotic drug, and has been applied in the fields of medicine and health care.

[0003] Patent CN111991384A discloses the application of protocatechuic acid and its compound protocatechuic acid in improving livestock and poultry performance. This invention relates to a new use of protocatechuic acid and its compound protocatechuic acid in the feed industry. This product can prevent and treat digestive tract diseases such as diarrhea and gastric ulcers, as well as immunosuppression in livestock and poultry caused by pathogenic Bacillus cereus, Bacillus thuringiensis, and Bacillus pumilus. In vitro and in vivo tests show that compound protocatechuic acid can kill pathogenic Bacillus cereus, Bacillus pumilus, and Bacillus thuringiensis, while also preventing and treating diarrhea, gastric ulcers, and intestinal hemorrhagic diseases caused by these three Bacillus species. The dosage is 12.5 mg / kg body weight or 200 g / ton of feed. The compound protocatechuic acid's ability to kill pathogenic Bacillus cereus provides a new technical approach for healthy livestock and poultry farming.

[0004] Since 2008, with the increasing application of protocatechuic acid in pharmaceuticals, feed, and environmental protection, the production and demand of protocatechuic acid have steadily increased, with an average annual growth rate of about 10%. In 2015, the global total production was approximately 250 tons, with a market capacity of about 125 million yuan. Currently, almost all domestic protocatechuic acid producers use chemical processes, with an estimated production cost of over 150,000 yuan / ton and a market price of 450,000-500,000 yuan / ton. The average profit margin can reach about 50%. However, the chemical synthesis of protocatechuic acid generates a large amount of waste, which contradicts my country's current environmental policies. Producers generally face significant environmental pressure, and some have already suspended production for rectification. Therefore, developing green and environmentally friendly production technologies is highly competitive and represents the direction for industrial development.

[0005] Patent CN104098465A discloses a process for extracting protocatechuic acid from *Artemisia argyi* (Yunnan-Guangxi), belonging to the field of traditional Chinese medicine extraction technology. This method involves the following steps to obtain protocatechuic acid monomers: water extraction, concentration, extraction, elution, and TLC purification. HPLC analysis determined that compound B is ≥99% protocatechuic acid, confirming it as a protocatechuic acid monomer. This patented process for extracting protocatechuic acid from *Artemisia argyi* solves problems such as low purity, small yield, and complex preparation processes. Through water extraction, concentration, extraction, and elution, it achieves a new process for extracting protocatechuic acid with high purity, a simple preparation process, and suitability for widespread application, meeting the demand for protocatechuic acid. However, this method suffers from drawbacks such as low extraction rate and high extraction cost of protocatechuic acid monomers.

[0006] Honeysuckle vine is the dried stem and branches of *Lonicera japonica* Thunb., a plant in the Caprifoliaceae family. It is sweet and cold in nature, possessing the effects of clearing heat and detoxifying, dispelling wind and unblocking meridians. It is used to treat febrile diseases, dysentery due to heat toxins, carbuncles, and sores. Honeysuckle vine mainly contains flavonoids, organic acids, and triterpenoids, exhibiting pharmacological activities such as antitumor, anti-inflammatory, antibacterial, immunomodulatory, and antipyretic effects. While there are existing reports on the presence of protocatechuic acid in honeysuckle vine, there are no reports on the direct extraction of protocatechuic acid from it. Therefore, how to effectively extract protocatechuic acid from honeysuckle vine is a key research topic worthy of study in this field.

[0007] Based on the above, it is necessary to develop an extraction process for protocatechuic acid from honeysuckle vine and to study the new functions and applications of protocatechuic acid in the aquaculture industry, so as to provide important basis and reference for the research project on the medicinal value of protocatechuic acid in animal breeding. Summary of the Invention

[0008] The present invention aims to solve the problems existing in the prior art and provide a method for preparing honeysuckle vine extract and its application.

[0009] The technical solution of this invention is as follows:

[0010] In one aspect, the present invention provides a method for preparing honeysuckle vine extract, wherein the preparation steps are as follows:

[0011] Step 1: Boil the raw honeysuckle vine in water, filter it to obtain the filtrate, and concentrate the filtrate to a concentration with a relative density of 1.01-1.10 at 60-80℃;

[0012] Step 2: Extract the concentrate obtained in Step 1 with an organic solvent, retaining the organic layer;

[0013] Step 3: Concentrate the organic layer obtained in Step 2 to dryness under reduced pressure, extract with hot water, filter, retain the aqueous layer, acidify the aqueous layer and ether extract, retain the ether layer, extract the ether layer with 5% sodium bicarbonate solution, and retain the sodium bicarbonate extract.

[0014] Step 4: Acidify the sodium bicarbonate extract obtained in Step 3, adjust the pH, concentrate and crystallize, and recrystallize with CHCl3 to obtain honeysuckle vine extract.

[0015] In step 1, the raw honeysuckle vine is decocted twice with water. The two decoctions are filtered separately and then combined to obtain the filtrate. For the first decoction, add 8-10 times the amount of water as the raw honeysuckle vine and decoct for 1-2 hours. For the second decoction, add 8-10 times the amount of water as the raw honeysuckle vine and decoct for 1-1.5 hours.

[0016] Preferably, in step 1, the relative density of the concentrate is 1.08-1.10, and after concentration, the pH of the concentrate is adjusted to 2-6; preferably, the pH is 4-5, and more preferably, the pH is 4.

[0017] In step 2, the organic solvent used is butanone or ethyl acetate, and the total amount of organic solvent used is 0.2-1 times the volume of the concentrate.

[0018] In step 3, the hot water temperature is 60-80℃, the total amount of hot water used is 5-6 times the volume of the concentrated residue, the water layer is acidified with 12mol / L concentrated hydrochloric acid, the pH value of the water layer is 5-6, the total amount of diethyl ether used is 0.2-1 times the volume of the water layer, and the total amount of sodium bicarbonate solution used is 0.2-1 times the volume of the ether layer.

[0019] In step 4, the sodium bicarbonate extract is acidified with 12 mol / L concentrated hydrochloric acid to adjust the pH of the sodium bicarbonate extract to 5-6.

[0020] Specifically, the preparation method steps are as follows:

[0021] Step 1: Add 8-10 times the amount of water to the raw honeysuckle vine and decoct for 1-2 hours. Filter and collect decoction I. Add 8-10 times the amount of water to the remaining residue and decoct for 1-1.5 hours. Filter and collect decoction II. Combine decoction I and decoction II to obtain a filtrate. The filtrate is coffee-colored. Concentrate the filtrate until the relative density of the concentrate is 1.08-1.10 at 60-80℃. Adjust the pH to 2-6.

[0022] Step 2: After cooling the concentrate obtained in Step 1, extract it with an organic solvent 3-5 times. The total amount of organic solvent used is 0.2-1 times the volume of the concentrate. Separate the water layer and the organic layer, and retain the organic layer.

[0023] Step 3: Concentrate the organic layer obtained in Step 2 to dryness under reduced pressure, dissolve it with hot water at 60-80℃, filter, discard the water-insoluble matter, and retain the aqueous layer. The total amount of hot water used should be 5-6 times the volume of the concentrated residue. Acidify the aqueous layer with 12mol / L concentrated hydrochloric acid to adjust the pH to 5-6, and perform ether extraction. The total amount of diethyl ether used should be 0.2-1 times the volume of the aqueous layer. Discard the aqueous layer and retain the ether layer. Extract the ether layer with 5% sodium bicarbonate solution. The total amount of sodium bicarbonate solution used should be 0.2-1 times the volume of the ether layer. Discard the ether layer and retain the sodium bicarbonate extract.

[0024] Step 4: Acidify the sodium bicarbonate extract obtained in Step 3 with 12 mol / L concentrated hydrochloric acid, adjust the pH to 5-6, concentrate under reduced pressure to obtain crystals, and recrystallize the crystals with chloroform (CHCl3) to obtain colorless needle-like honeysuckle vine extract, namely protocatechuic acid (3,4-dihydroxybenzoic acid) crystals.

[0025] In another aspect, the present invention provides the application of the honeysuckle vine extract as a feed additive in livestock and poultry farming.

[0026] In another aspect, the present invention provides the use of the honeysuckle vine extract in the preparation of a combination drug.

[0027] Preferably, the combined drug is a combination of honeysuckle vine extract and antibiotic.

[0028] More preferably, the antibiotic is florfenicol.

[0029] The protocatechuic acid of this invention is prepared from the natural plant honeysuckle vine. Honeysuckle vine has a pungent, sweet, and cold nature, and enters the lung and stomach meridians. It has the effects of clearing heat, detoxifying, and unblocking the meridians. Modern medical research has proven that protocatechuic acid has a strong antibacterial effect against Gram-negative bacteria, can prevent the occurrence of bacterial disease infections, and has anti-inflammatory, antioxidant, and immune-regulating effects, thereby achieving the purpose of replacing the use of feed antibiotics.

[0030] The beneficial effects of this invention are:

[0031] 1. The protocatechuic acid of this invention is prepared from the natural plant honeysuckle vine. It has a mild effect and, compared with antibiotics, hormones, and chemically synthesized drugs, can prevent drug residues in the body, has no toxic side effects, and is safe and reliable.

[0032] 2. The honeysuckle vine extract of the present invention has significant antibacterial and anti-inflammatory effects with a long duration of action, and can effectively inhibit the activity of Escherichia coli and Salmonella typhimurium.

[0033] 3. The protocatechuic acid extracted in this invention has a synergistic effect when used in combination with antibiotics, which can significantly reduce the clinical dosage of antibiotics. 1.56 mg / ml of honeysuckle vine extract and 1.24 mg / ml of florfenicol can completely inhibit and kill Escherichia coli and Salmonella typhimurium.

[0034] 3. The honeysuckle vine extraction method of the present invention can extract protocatechuic acid with a purity of over 95% and an extraction rate of 0.01%-0.1%. The honeysuckle vine extraction method of the present invention can extract high-purity protocatechuic acid from honeysuckle vine, which greatly saves medicinal materials and avoids waste.

[0035] 4. The honeysuckle vine extract of the present invention can be used as a feed additive by mixing it directly into feed at the breeding end, or by combining it with other drugs or feed additives, or by adding it to various types of feed.

[0036] 5. The preparation process of this invention is simple, easy to use, the raw materials are readily available, the price is low, and it is green and environmentally friendly. Attached Figure Description

[0037] Figure 1 This is a process flow diagram of the honeysuckle vine extraction method in this invention;

[0038] Figure 2 The chromatographic peak of protocatechuic acid standard;

[0039] Figure 3 The chromatographic peaks of the honeysuckle vine extract obtained by the method of this invention are compared. Detailed Implementation

[0040] The technical solution of the present invention will be further described below through specific embodiments.

[0041] Example 1: Preparation of Honeysuckle Vine Extract

[0042] (1) Add 100g of raw honeysuckle vine to water and decoct twice. Add 8 times the amount of water for the first decoction and decoct for 1.5 hours. Add 8 times the amount of water for the second decoction and decoct for 1 hour. Filter the decoctions with a 200-mesh filter cloth. Combine the decoctions to obtain the filtrate. Concentrate the filtrate to a concentration with a relative density of about 1.10 at 60℃ and adjust the pH to 4.

[0043] (2) After cooling the concentrate, extract it with ethyl acetate 3-5 times. The total amount of ethyl acetate used is 0.2 times the volume of the concentrate, and the ester layer is retained.

[0044] (3) Concentrate the ester layer to dryness under reduced pressure, dissolve it in 60℃ hot water (total volume of hot water is 5 times the volume of the concentrated residue), filter, discard the water-insoluble matter, and retain the aqueous layer. Acidify the aqueous layer with 12mol / L concentrated hydrochloric acid to adjust the pH to 5, and perform ether extraction (total volume of diethyl ether is 0.2 times the volume of the aqueous layer). Discard the aqueous layer and retain the ether layer. Extract the ether layer with 5% sodium bicarbonate solution (total volume of sodium bicarbonate solution is 0.2 times the volume of the ether layer). Discard the ether layer and retain the sodium bicarbonate extract.

[0045] (4) The sodium bicarbonate extract was acidified with 12 mol / L concentrated hydrochloric acid, the pH was adjusted to 5, and the solution was concentrated and crystallized. The resulting crystals were then recrystallized with CHCl3 to obtain colorless needle-like compound 1. The melting point of compound 1 was 198-200℃. HPLC analysis determined that compound 1 was 95.5% protocatechuic acid (3,4-dihydroxybenzoic acid), and the extraction rate of compound 1 was 0.1%.

[0046] Example 2: Preparation of Honeysuckle Vine Extract

[0047] (1) Add 100g of raw honeysuckle vine to water and decoct twice. Add 10 times the amount of water for the first decoction and decoct for 2.0h. Add 10 times the amount of water for the second decoction and decoct for 1.5h. Filter the decoctions with a 200-mesh filter cloth. Combine the decoctions to obtain the filtrate. Concentrate the filtrate to a concentration with a relative density of about 1.09 at 80℃ and adjust the pH to 6.

[0048] (2) After cooling the concentrate, extract it with ethyl acetate 3-5 times. The total amount of ethyl acetate used is 1 times the volume of the concentrate, and the ester layer is retained.

[0049] (3) Concentrate the ester layer to dryness under reduced pressure, dissolve it in 80℃ hot water (total volume of hot water is 6 times the volume of the concentrated residue), filter, discard the water-insoluble matter, and retain the aqueous layer. Acidify the aqueous layer with 12mol / L concentrated hydrochloric acid, adjust the pH to 6, and perform ether extraction (total volume of diethyl ether is 1 times the volume of the aqueous layer). Discard the aqueous layer and retain the ether layer. Extract the ether layer with 5% sodium bicarbonate solution (total volume of sodium bicarbonate solution is 1 times the volume of the ether layer). Discard the ether layer and retain the sodium bicarbonate extract.

[0050] (4) The sodium bicarbonate extract was acidified with 12 mol / L concentrated hydrochloric acid, the pH was adjusted to 6, and the solution was concentrated and crystallized. The resulting crystals were then recrystallized with CHCl3 to obtain colorless needle-like compound 2. The melting point of compound 2 was 198-200℃. HPLC analysis determined that compound 2 was 95.1% protocatechuic acid (3,4-dihydroxybenzoic acid), and the extraction rate of compound 2 was 0.08%.

[0051] Example 3: Preparation of Honeysuckle Vine Extract

[0052] (1) Add 100g of raw honeysuckle vine to water and decoct twice. Add 9 times the amount of water for the first decoction and decoct for 1.5h. Add 9 times the amount of water for the second decoction and decoct for 1h. Filter the two decoctions with a 200-mesh filter cloth. Combine the decoctions to obtain the filtrate. Concentrate the filtrate to a concentration with a relative density of about 1.06 at 70℃ and adjust the pH to 5.

[0053] (2) After cooling, extract with ethyl acetate 3-5 times. The total amount of ethyl acetate used is 0.5 times the volume of the concentrate. The ester layer is retained.

[0054] (3) Concentrate the ester layer to dryness under reduced pressure, dissolve it in 70°C hot water (5.5 times the volume of the concentrated residue), filter, discard the water-insoluble matter, and retain the aqueous layer. Acidify the aqueous layer with 12mol / L concentrated hydrochloric acid to adjust the pH to 5.5, and extract with ether (0.5 times the volume of the aqueous layer). Discard the aqueous layer and retain the ether layer. Extract the ether layer with 5% sodium bicarbonate solution (0.5 times the volume of the ether layer). Discard the ether layer and retain the sodium bicarbonate extract.

[0055] (4) The sodium bicarbonate extract was acidified with 12 mol / L concentrated hydrochloric acid, the pH was adjusted to 5.5, and the solution was concentrated and crystallized. The resulting crystals were then recrystallized with CHCl3 to obtain colorless needle-like compound 3. The melting point of compound 3 was 198-200℃. HPLC analysis determined that compound 3 was 95.3% protocatechuic acid (3,4-dihydroxybenzoic acid), and the extraction rate of compound 3 was 0.09%.

[0056] Example 4: Preparation of Honeysuckle Vine Extract

[0057] (1) Add 100g of raw honeysuckle vine to water and decoct twice. Add 9 times the amount of water to the first decoction and decoct for 1 hour. Add 9 times the amount of water to the second decoction and decoct for 1 hour. Filter the decoctions with a 200-mesh filter cloth. Combine the decoctions to obtain the filtrate. Concentrate the filtrate to a concentration with a relative density of about 1.06 at 70℃ and adjust the pH to 2.

[0058] (2) After cooling, extract with butanone 3-5 times. The total amount of butanone used is 0.5 times the volume of the concentrated liquid, and the organic layer is retained.

[0059] (3) Concentrate the organic layer to dryness under reduced pressure, dissolve it in 70℃ hot water (total volume of hot water is 5.5 times the volume of the concentrated residue), filter, discard the water-insoluble matter, and retain the aqueous layer. Acidify the aqueous layer with 12mol / L concentrated hydrochloric acid, adjust the pH to 5.5, and extract with ether (total volume of diethyl ether is 0.5 times the volume of the aqueous layer). Discard the aqueous layer and retain the ether layer. Extract the ether layer with 5% sodium bicarbonate solution (total volume of sodium bicarbonate solution is 0.5 times the volume of the ether layer). Discard the ether layer and retain the sodium bicarbonate extract.

[0060] (4) The sodium bicarbonate extract was acidified with 12 mol / L concentrated hydrochloric acid, the pH was adjusted to 5.5, and the solution was concentrated and crystallized. The resulting crystals were then recrystallized with CHCl3 to obtain colorless needle-like 3,4-dihydroxybenzoic acid compound 4. The melting point of compound 4 was 198-200℃. HPLC analysis determined that compound 4 was 94.5% protocatechuic acid (3,4-dihydroxybenzoic acid), and the extraction rate of compound 4 was 0.06%.

[0061] Comparative Example 1: Preparation of Honeysuckle Vine Extract

[0062] All other steps and conditions were the same as in Example 1, except that the reagents used for recrystallization were DMF, chlorobenzene, toluene, and petroleum ether, respectively. The resulting compounds were 95.3% protocatechuic acid (3,4-dihydroxybenzoic acid), 94.7% protocatechuic acid (3,4-dihydroxybenzoic acid), 95.0% protocatechuic acid (3,4-dihydroxybenzoic acid), and 93.3% protocatechuic acid (3,4-dihydroxybenzoic acid), respectively, with extraction rates of 0.08%, 0.07%, 0.09%, and 0.08%, respectively.

[0063] Comparative Example 2: Preparation of Honeysuckle Vine Extract

[0064] Steps (1) and (2) are the same as in Example 1, except that steps (3) and (4) are adjusted as follows:

[0065] Step (3) Elution: Take the ester layer silica gel obtained in step (2), mix it well, and load it onto a silica gel dry column. Use a solution prepared in the ratio of chloroform-methanol-water of 65:35:10 as the eluent for gradient elution. Collect the lower aqueous phase of the eluent for each gradient.

[0066] Step (4) TLC preparation and purification: The aqueous phase obtained in the previous step was identified by TLC. The lower aqueous phase in which the target component was detected was taken and a solution prepared with chloroform:acetone:methanol in a ratio of 6:1:1 was used as the developing solvent for TLC preparation. The compound was finally obtained. The compound was crystallized and dried. The above compound was detected by HPLC. The obtained compound was 99% protocatechuic acid (3,4-dihydroxybenzoic acid). The extraction rate of the obtained compound was only 0.01%.

[0067] Structure determination of compound in Experimental Example 1

[0068] HPLC (High Performance Liquid Chromatography)

[0069] 1. Instruments and reagents

[0070] High performance liquid chromatograph: Shimadzu LC-6A, Japan; High performance liquid chromatographic column: Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Ф4.6×250mm, packing material: Nucleosil 7C18, UV detection at 260nm.

[0071] 2. Method

[0072] (1) Chromatographic conditions: high performance liquid chromatography column Nucleosil 7C18 (Ф4.6×250mm), mobile phase is methanol-1% glacial acetic acid solution (19:81, v / v), flow rate is 1ml / min, column temperature is room temperature, detection wavelength is 260nm, and the theoretical plate number of protocatechuic acid peak should not be lower than 4000.

[0073] (2) Preparation of reference solution and test solution:

[0074] Accurately weigh 3.0 mg of protocatechuic acid reference standard and place it in a 10 ml volumetric flask. Dissolve and dilute to the mark with 70% ethanol, and shake well to obtain a 0.30 mg / ml reference standard stock solution. Transfer 1 ml of the above stock solution to a 10 ml volumetric flask, dissolve and dilute to the mark with 70% ethanol, and shake well to obtain a 0.030 mg / ml reference standard solution.

[0075] Accurately weigh 30 mg of protocatechuic acid (i.e., compounds 1-4 prepared in Examples 1-4), place them in separate 50 ml volumetric flasks, dissolve and dilute to the mark with 70% ethanol, and shake well to obtain the test sample stock solution. Transfer 1 ml of the above stock solution to a 25 ml volumetric flask, dissolve and dilute to the mark with 70% ethanol, and shake well to obtain a 0.024 mg / ml test sample solution 1-4.

[0076] (3) Precision test: 10 μl of each of the reference solution and the test solution 1-4 were injected into the high performance liquid chromatograph. After comparing the chromatographic peaks with those of the reference solution, it was determined that compounds 1-4 were all protocatechuic acid, and the content of each protocatechuic acid in compounds 1-4 was determined.

[0077] 3. Results: HPLC analysis showed that compounds 1-4 had the same retention time as the protocatechuic acid standard and were white crystals with a melting point of 198-200℃. Based on the HPLC data analysis, compounds 1-4 were determined to be protocatechuic acid, i.e., 3,4-dihydroxybenzoic acid.

[0078] Example 2: Antibacterial test of honeysuckle vine extract

[0079] Experimental Method: The "microplate" method was used. A 96-well cell culture plate was prepared, with 100 μl of culture medium added to each well. 100 μl of the honeysuckle vine extract solution of this invention was added to the first well, and after thorough mixing, 100 μl was transferred to the second well. This serial dilution was repeated until the 12th well, from which 100 μl was discarded. Then, 10 μl of the diluted bacterial solution was added to each well. After incubation at 37°C for 18 hours, the turbidity of each well was visually observed. Turbidity indicated bacterial growth; the concentration of the drug in the last well that remained clear was the MIC value. The solutions from each well were streaked onto the plate and incubated at 37°C for another 18-24 hours. The drug dilution corresponding to the absence of bacterial growth on the plate was the MBC value. The "microplate" method requires parallel controls, and the positive control must show bacterial growth while the control group must show no bacterial growth to determine the MIC and MBC values. The antibacterial test results of compounds 1-4 are shown in Table 1.

[0080] Table 1. MIC and MBC values ​​of single-drug therapy

[0081]

[0082] Conclusion: Based on the comprehensive analysis of the MIC and MBC values ​​of compounds 1-4 in Table 1, compound 1 (prepared in Example 1) showed significantly higher antibacterial effects against Escherichia coli and Salmonella typhimurium than compounds 2-4 (prepared in Examples 2-4).

[0083] Example 3: Antibacterial test of honeysuckle vine extract in combination with chemical drugs

[0084] Experimental Method: Compound 1 (prepared in Example 1), exhibiting the best antibacterial effect, was selected for the subsequent antibacterial test. The drug stock solution was diluted twice with bacterial culture medium to a specific mass concentration. 20 μl of serially diluted florfenicol was added to eight wells of a 96-well plate at concentrations of 0.0625 mg / ml, 0.125 mg / ml, 0.25 mg / ml, 0.50 mg / ml, 1.00 mg / ml, 2.00 mg / ml, 4.00 mg / ml, and 8.00 mg / ml. Similarly, 20 μl of serially diluted compound 1 was added to the remaining eight wells of the 96-well plate at concentrations of 0.10 mg / ml, 0.20 mg / ml, 0.39 mg / ml, 0.78 mg / ml, 1.56 mg / ml, 3.12 mg / ml, 6.24 mg / ml, and 12.48 mg / ml. For a 96-well plate, 20 μl of chlorogenic acid, serially diluted to 0.00625 mg / ml, 0.0125 mg / ml, 0.025 mg / ml, 0.05 mg / ml, 0.10 mg / ml, 0.20 mg / ml, 0.40 mg / ml, and 0.80 mg / ml, was added sequentially to 8 wells of the 96-well plate. Similarly, 20 μl of compound 1, serially diluted to 0.10 mg / ml, 0.20 mg / ml, 0.39 mg / ml, 0.78 mg / ml, 1.56 mg / ml, 3.12 mg / ml, 6.24 mg / ml, and 12.48 mg / ml, was added sequentially to the other 8 wells of the 96-well plate. After thoroughly mixing the drug solution in each well, 20 μl of the mixture was discarded. Finally, 200 μl of diluted bacterial suspension was added to each well of the two 96-well plates. After incubation at 37°C for 18 hours, the turbidity of each well was visually observed. Turbidity indicates bacterial growth; if the plate is clear, the drug concentration in the last clear well is the MIC value. Each well solution was streaked onto the plate and incubated at 37°C for another 18-24 hours. The drug dilution corresponding to the absence of bacterial growth on the plate is the MBC value. The "microplate" method requires parallel controls, with the positive control showing bacterial growth and the control group showing no bacterial growth, before the MIC and MBC values ​​can be determined. The experimental results are shown in Table 2.

[0085] Table 2. MIC and MBC values ​​of single drugs

[0086]

[0087] Calculation of FICI and efficacy evaluation criteria: FICI value = (MIC of drug A in combined action / MIC of drug A in single action) + (MIC of drug B in combined action / MIC of drug B in single action). When FICI ≤ 0.5, it is determined that the two drugs have a synergistic effect; when 0.5 < FICI < 4.0, it is determined to be an indifferent effect; when FICI ≥ 4.0, it is determined to be an antagonistic effect. The test results are shown in Table 3.

[0088] Table 3 Measured values of MIC and MBC of combined medication

[0089]

[0090] Conclusion: The combination of protocatechuic acid and florfenicol can synergistically enhance the efficacy to inhibit the growth of Escherichia coli and Salmonella typhimurium; the optimal formulation ratio is protocatechuic acid 1.56 mg / ml + florfenicol 1.24 mg / ml, which can completely inhibit and kill Escherichia coli and Salmonella typhimurium, while the effect of the combination of chlorogenic acid and florfenicol is not significant.

[0091] Test Example 4 Clinical therapeutic effect test of Lonicerae Japonicae Caulis extract on LPS-induced endometritis in mice

[0092] Test method: Compound 1 (prepared in Example 1) was selected for the following anti-inflammatory test. Fifty mice were randomly divided into 5 groups: control group, inflammation model group, and treatment groups (15, 30, 60 mg / kg), with 10 mice in each group. Mice in the control group were perfused with 50 μl of normal saline through the vagina with a micro-syringe, mice in the inflammation model group were perfused with 50 μl of inflammation inducer LPS (1 mg / kg), and mice in the treatment groups were intraperitoneally injected with 15, 30 and 60 mg / kg of Compound 1 respectively 1 h before being perfused with the inflammation inducer. 24 h after injection, all mice were sacrificed by cervical dislocation. Uterine tissues were collected, the content of inflammatory factor NO was detected with a NO kit, and the contents of inflammatory factors tumor necrosis factor-α (TNF-α) and IL-6 were detected by ELISA. The test results are shown in Table 4.

[0093] Table 4 Expression levels of inflammatory factors in mouse uterus (n=10, mean ± standard deviation)

[0094] control group 7.90±0.97 185.00±1.92 24.82±1.11 Inflammation model group 35.55±0.17** 285.00±1.02** 80.63±0.74** Compound 115 mg / kg 27.65±0.68## 232.14±0.71## 45.39±0.49## Compound 130 mg / kg 25.68±0.46## 210.24±0.79## 39.33±1.52## Compound 160 mg / kg 19.52±1.53## 201.62±1.78## 28.57±2.39##

[0095] Note: Compared with the control group, ** represents extremely significant difference (P<0.01), and * represents significant difference (P<0.05). Compared with the inflammation model group, ## represents extremely significant difference (P<0.01), and # represents significant difference (P<0.05).

[0096] Results: Compared with the control group, the levels of NO, TNF-α and IL-6 in the endometrial tissue of mice in the inflammation model group were significantly increased (P<0.01); compared with the inflammation model group, the levels of inflammatory cytokines NO, TNF-α and IL-6 in the endometrial tissue of mice in the protocatechuic acid treatment group were significantly decreased (P<0.01) in a dose-dependent manner.

[0097] Conclusion: Protocatechuic acid extracted from honeysuckle vine using the method of this invention has a protective effect against LPS-induced endometritis in mice by inhibiting the NF-κB signaling pathway and the production of inflammatory cytokines.

[0098] While specific embodiments of the present invention have been described above, those skilled in the art should understand that the specific embodiments described are merely illustrative and not intended to limit the scope of the present invention. Equivalent modifications and variations made by those skilled in the art in accordance with the spirit of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing honeysuckle vine extract, characterized in that: The preparation steps are as follows: Step 1: Boil the raw honeysuckle vine in water, filter it to obtain the filtrate, concentrate the filtrate to a concentration with a relative density of 1.08-1.10 at 60-80℃, and adjust the pH of the concentration to 4-5 after concentration. Step 2: Extract the concentrate obtained in Step 1 with an organic solvent, retaining the organic layer; Step 3: Concentrate the organic layer obtained in Step 2 under reduced pressure to dryness, extract with hot water, filter, retain the aqueous layer, acidify the aqueous layer and ether extract, retain the ether layer, extract the ether layer with 5% sodium bicarbonate solution, and retain the sodium bicarbonate extract. Step 4: Acidify the sodium bicarbonate extract obtained in Step 3, adjust the pH, concentrate and crystallize, and recrystallize with CHCl3 to obtain honeysuckle vine extract. In step 1, the raw honeysuckle vine is decocted twice with water. The two decoctions are filtered separately and then combined to obtain the filtrate. For the first decoction, add 8-10 times the amount of water as the raw honeysuckle vine and decoct for 1-2 hours. For the second decoction, add 8-10 times the amount of water as the raw honeysuckle vine and decoct for 1-1.5 hours. In step 2, the organic solvent used is butanone or ethyl acetate, and the total amount of organic solvent used is 0.2-1 times the volume of the concentrate. In step 3, the hot water temperature is 60-80℃, the total amount of hot water used is 5-6 times the volume of the concentrated residue, the water layer is acidified with 12mol / L concentrated hydrochloric acid, the pH value of the water layer is 5-6, the total amount of ether used is 0.2-1 times the volume of the water layer, and the total amount of sodium bicarbonate solution used is 0.2-1 times the volume of the ether layer. In step 4, the sodium bicarbonate extract is acidified with 12 mol / L concentrated hydrochloric acid to adjust the pH of the sodium bicarbonate extract to 5-6. The active ingredient in honeysuckle vine extract is 3,4-dihydroxybenzoic acid, and the purity of 3,4-dihydroxybenzoic acid in honeysuckle vine extract is ≥95%; the extraction rate of 3,4-dihydroxybenzoic acid in honeysuckle vine is 0.01%-0.1%.

2. The method for preparing honeysuckle vine extract according to claim 1, characterized in that, The honeysuckle vine extract is used to prepare a combination drug, which is a combination of honeysuckle vine extract and an antibiotic; the combination drug is 1.56 mg / ml of honeysuckle vine extract and 1.24 mg / ml of florfenicol.

3. The method for preparing honeysuckle vine extract according to claim 1, characterized in that, The honeysuckle vine extract can be used as a feed additive in livestock and poultry farming.

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