Magnolol inclusion compound and application of preparation thereof in inhibiting vibrio

Abstract

The invention belongs to the technical field of natural plant active products, and particularly relates to a magnolol clathrate compound for inhibiting vibrio in aquaculture and a preparation and application of the magnolol clathrate compound. Magnolol is high-purity magnolol obtained by separating and purifying an active component extracted from a natural plant raw material mangnolia officinalis through a subcritical process. Molecular clathration is carried out on magnolol by adopting a molecular clathration technology to prepare a magnolol clathrate compound, the magnolol clathrate compound is further prepared into different dosage forms such as powder, granules and a liquid preparation to obtain an anti-vibrio preparation product, and tests prove that the anti-vibrio effect of the preparation product is remarkable. The raw materials are low in price and easy to obtain, the magnolol extraction process is simple, green and environmentally friendly, magnolol is clathrated through the molecular clathration technology, the water solubility and oral utilization rate of magnolol are increased, the prepared preparation can be added into feed, the use of antibiotics is reduced, healthy development of green fishery is promoted, and the economic benefit is increased. And a foundation is laid for development of antibiotic-free aquatic natural plant feed additive system products.

Description

Technical Field

[0001] The invention relates to the technical application field of natural plant active products, and in particular to a magnolol inclusion compound used for inhibiting Vibrio in aquaculture, and a preparation and application thereof. Background Art

[0002] Magnolia officinalis belongs to the Magnoliaceae family and Magnolia genus. Its bark is a traditional Chinese medicinal material and is also a natural plant feed raw material catalog variety. Magnolia officinalis is the main active ingredient of Magnolia officinalis. The molecular formula of Magnolia officinalis is C 18 H 18 O 2 (As shown in the following formula), the molecular weight is 266.32, it is colorless needle-shaped crystals, it is easily soluble in organic solvents such as chloroform, acetone, ether, slightly soluble in water, and its melting point is 102°C. Magnolol is known to have multiple pharmacological effects such as antibacterial, anti-tumor, antioxidant, and anti-ulcer. Studies have shown that magnolol has significant antibacterial activity against a variety of Gram-positive bacteria, acid-resistant bacteria, yeast-like fungi, and filamentous fungi. It has strong inhibitory activity against bacteria such as Escherichia coli, Shigella dysenteriae, Staphylococcus aureus, and a variety of plant pathogenic fungi, and does not affect the survival of normal intestinal flora. At present, there are many reports on the antibacterial application of magnolia bark phenol in poultry, livestock, etc., and there are few reports on the bactericidal activity against aquatic pathogens. Guo Bingyu et al. conducted an antibacterial experiment on magnolia bark phenol against Vibrio parahaemolyticus (a more pathogenic Vibrio) in the literature on the antibacterial effect of magnolia bark phenol on several pathogens. The results showed that magnolia bark phenol has a good antibacterial effect on Vibrio parahaemolyticus.

[0003]

[0004] Natural plants contain many highly effective, safe and environmentally friendly antibacterial ingredients, which are the main resources for developing alternative antibiotic products for aquatic animals. Developing anti-aquatic pathogens through natural plants can replace the use of antibiotics. At the same time, Magnolia officinalis has many origins, and the raw materials are easy to obtain and cheap. The active ingredient magnolol has a definite antibacterial effect, which is in line with the development trend of antibiotic-free farming in my country.

[0005] Due to the poor solubility of magnolol in aqueous solution, some researchers have added Tween-80 and DMSO as cosolvents, but this only increased the solubility of magnolol to a certain extent, restricting its wider application in aquaculture. Therefore, in future research, by using solubilization, eutectic and other technologies to improve the solubility of magnolol, the solubility of magnolol in water can be improved and the antibacterial ability can be enhanced, expanding its application space in aquaculture.

[0006] Vibrio parahaemolyticus is a Gram-negative bacterium of the Vibrio family. It is a member of the pathogenic Vibrio. It is mostly arc-shaped, rod-shaped or oval-shaped under an electron microscope. It is widely distributed in salty water bodies such as seawater and saltwater lakes. Vibrio has a wide range of pathogenicity to marine aquaculture animals and can cause infectious diseases in a variety of fish, shrimps, crabs, shellfish, etc. It is the main pathogen causing acute hepatopancreatic necrosis syndrome in white shrimp. The optimal growth temperature of this pathogen is 37℃, so rainy days in summer are the high-incidence season for such diseases. Vibrio is extremely harmful to white shrimp. Once infected with shrimp, the mortality rate can be as high as 80% or even 100%, which will cause a large number of sick shrimp deaths in a short period of time, seriously affecting the healthy development of white shrimp farming in my country. Therefore, the screening of magnolol, a natural plant feed raw material, and after being processed by molecular inclusion technology, can effectively reduce the occurrence of vibrio diseases, which is of great significance for the healthy breeding of marine shrimp and the safety of animal food.

[0007] In fishery production, the regular use of disinfectants and microecological preparations is the most common external means to reduce the abnormal reproduction of Vibrio in water bodies, while oral chemical antimicrobial drugs are a common internal means of preventing and treating Vibrio disease. However, the long-term and large-scale use of chemical antimicrobial drugs can easily lead to drug residues in the aquaculture environment and in aquatic animals, as well as the emergence of drug-resistant bacteria, posing hidden dangers to aquaculture disease prevention and control and aquatic animal-derived food safety. At the same time, the large-dose use of chemical drugs will cause damage to the algae phase of the water body. In addition, farmers are increasingly aware of the hazards caused by the excessive use of chemical antimicrobial drugs, which has also prompted people to look for more environmentally friendly and safer solutions. Therefore, seeking a safer, more effective and low-cost ingredient to inhibit Vibrio in water bodies has important economic and social value.

[0008] Among the patents currently applied for, there are those that use Chinese medicine compositions or Chinese medicine extracts to inhibit Vibrio, those that use single extract ingredients such as emodin and berberine to inhibit Vibrio, and those that use natural active ingredients in combination with antibiotics to inhibit Vibrio, but there is no patent for magnolol to inhibit Vibrio. The present invention combines existing patents and literature, studies the characteristics of magnolol, optimizes the extraction and purification process, prepares high-purity magnolol, and on this basis, includes magnolol through molecular inclusion technology to increase its water solubility and bioavailability. The prepared preparation has passed in vivo and in vitro Vibrio killing experiments, and has obvious Vibrio killing effects, providing a new means for green breeding. Summary of the invention

[0009] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of the present invention is to provide a magnolol inclusion compound and its preparation for use in inhibiting Vibrio. Magnolia officinalis is extracted from magnolia officinalis by a subcritical process, and magnolia officinalis is crystallized and purified. In order to increase the water solubility and bioavailability of magnolia officinalis, the present invention adopts a macromolecular inclusion technology, and magnolia officinalis is included in the macromolecule, thereby improving the solubility and bioavailability of magnolia officinalis.

[0010] On the basis of the antibacterial effect of magnolol, the present invention discovered the antibacterial activity of magnolol against Vibrio, laying a foundation for the development of a series of green aquatic feed additive products.

[0011] The present invention is achieved through the following technical solutions:

[0012] A magnolol inclusion compound, wherein the inclusion compound is prepared from magnolol and hydroxypropyl-β-cyclodextrin, and the specific preparation method comprises the following steps:

[0013] (1) dissolving hydroxypropyl-β-cyclodextrin powder in water, and heating to dissolve to prepare a saturated aqueous solution;

[0014] (2) dissolving magnolol with 1 to 10 times of 70 to 90% ethanol, and slowly adding it to 5 to 10 times of saturated solution of hydroxypropyl-β-cyclodextrin under stirring;

[0015] (3) controlling the reaction temperature to 15 to 30° C., stirring the reaction at a stirring speed of 100 to 500 r / min for 1 to 5 hours, and standing for 12 to 24 hours;

[0016] (4) Filter out the white crystals, wash the residue 1 to 5 times with 5 to 10 times 70 to 95% ethanol, and dry in an oven at 60°C;

[0017] (5) Moisture content was controlled within 3%, and the mixture was crushed and passed through a 60-mesh sieve to obtain magnolol-β-cyclodextrin inclusion complex.

[0018] Furthermore, the magnolol is extracted and purified from magnolia bark by subcritical fluid technology, and the specific extraction and purification method comprises the following steps:

[0019] (1) Pretreatment: Magnolia officinalis bark is crushed into 20-50 meshes to obtain Magnolia officinalis granular powder, the Magnolia officinalis powder is put into a 200-mesh silk bag, and put into an extraction tank, the equipment is preheated to 60-75°C, and the vacuum pump is started to reduce the pressure in the extraction tank and the separation tank to 0-0.10 MPa;

[0020] (2) Extraction: Open the solvent injection valve of the extraction tank, inject an organic solvent (one of propane, butane, and liquefied petroleum gas) until the silk bag is submerged in the solvent and the liquid level is 3 to 5 cm higher than the silk bag. Turn on the hot water circulation system pump, maintain the extraction temperature at 30 to 75°C and the extraction pressure at 0.2 to 0.8 MPa for extraction for 30 to 90 minutes to obtain an oil-soluble mixture;

[0021] (3) desolventizing and separating: opening the sample outlet valve of the extraction tank and the sample inlet valve of the separation tank, after the oil-soluble mixture is pressed from the extraction tank into the separation tank, starting the compressor and the condensation system, heating, pressurizing and desolventizing the oil-soluble mixture, and when the pressure of the separation tank drops to 0.10-0.20 MPa, starting the vacuum pump and linking with the compressor to accelerate desolventizing and separating, thereby obtaining an organic solvent and a crude extract of Magnolia officinalis;

[0022] (4) Collecting the crude extract of Magnolia officinalis: the organic solvent enters the storage tank for recycling, the discharge valve of the separation tank is opened, the crude magnolol is collected, and the above extraction is repeated 3 to 5 times;

[0023] (5) Initial purification of magnolol: the crude extract was dissolved in 8-10 times the amount of 75% ethanol, heated to 40-50°C and filtered after being fully dissolved. The filtrate was separated by membrane separation technology, and the components with molecular weight cutoff less than 3000D were ultrafiltered, and then concentrated by nanofiltration membrane with molecular weight less than 200D. After concentration, the crude product was cooled and crystallized to obtain a crystalline crude product.

[0024] (6) Purification of magnolol: The crude crystalline product is dissolved in 2 to 5 times the amount of anhydrous ethanol, heated to 40 to 50°C until fully dissolved and then filtered. The filtrate is concentrated and cooled to 2 to 8°C to crystallize, and vacuum dried at 30 to 60°C to obtain the refined magnolol. The magnolol content is detected by HPLC.

[0025] Furthermore, the preparation of the magnolol inclusion compound is prepared by adding a synergist, a stabilizer and an excipient to the magnolol inclusion compound, wherein the mass ratio of the magnolol inclusion compound, the synergist, the stabilizer and the excipient is 5-10:2-5:1-10:70-90, and further, the mass ratio of the magnolol inclusion compound, the synergist, the stabilizer and the excipient is 10:5:10:75.

[0026] Furthermore, the synergist is one or more of dipotassium hydrogen phosphate, disodium hydrogen phosphate, sodium acetate, and potassium glycyrrhizinate; the stabilizer is one or more of β-cyclodextrin, sodium carboxymethyl cellulose, PEG6000, K30, sodium EDTA-2, and Tween-80; and the auxiliary material is one or more of glucose, starch, and an anti-caking agent.

[0027] Furthermore, the dosage form of the magnolol inclusion compound preparation is powder, granule or oral liquid preparation.

[0028] Furthermore, the present invention also discloses the application of the magnolol inclusion compound preparation in inhibiting Vibrio in aquaculture.

[0029] Furthermore, the magnolol inclusion compound preparation can effectively inhibit and kill Vibrio parahaemolyticus in vitro, with a minimum bactericidal concentration (MBC) of 25.6 μg / ml and a minimum inhibitory concentration (MIC) of 12.8 μg / ml.

[0030] Furthermore, the magnolol inclusion compound preparation can effectively inhibit and kill Vibrio in vivo, and 200g of the preparation mixed with 40-80kg of feed can effectively eliminate Vibrio in the intestine and hepatopancreas of white shrimp.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] 1. The present invention extracts magnolol from the natural plant raw material magnolia bark through a subcritical process, and then further separates and purifies to obtain high-purity magnolol, and finally adopts a molecular inclusion technology to include magnolol, thereby improving the solubility and bioavailability of magnolol. On this basis, different dosage forms such as powders, granules, and oral liquids are prepared to obtain magnolol inclusion compound preparation products. Through in vitro and in vivo tests on Vibrio, it is proved that the preparation process is stable, has a definite inhibitory and killing effect on Vibrio, and has a significant anti-Vibrio effect.

[0033] 2. The Magnolia officinalis medicinal material used in the product of the present invention is a natural plant feed raw material, which is green, environmentally friendly, safe and low-cost. The extraction process of magnolia officinalis phenol is simple and easy to industrialize. The prepared preparation can be added to feed. The promotion of the preparation can effectively reduce the use of chemical drugs and antibiotics, greatly reduce the drug resistance and drug resistance of aquatic animals, reduce the cost of medication, create greater social and economic benefits for the development of green breeding industry, and lay the foundation for the development of antibiotic-free aquatic natural plant feed additive system products, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a photo of the refined product of magnolia bark phenol according to Example 2 of the present invention.

[0035] Figure 2 This is the HPLC spectrum of the refined magnolia phenol product of Example 2 of the present invention, wherein the chromatographic conditions are: the filler is octadecylsilane bonded silica gel, the mobile phase is methanol: water (78:22), and the detection wavelength is 294 nm.

[0036] Figure 3 This is a comparison diagram of the antibacterial effect of the magnolol inclusion compound preparation of Example 2 of the present invention on Vibrio hepatopancreas; wherein, the left figure is a control sample of the hepatopancreas of white shrimp, and the right figure is a diagram of the antibacterial effect of the magnolol inclusion compound preparation on Vibrio hepatopancreas of white shrimp.

[0037] Figure 4 This is a comparison diagram of the antibacterial effect of the magnolol inclusion compound preparation of Example 2 of the present invention on intestinal Vibrio; wherein, the left figure is a control sample of the intestinal tract of white shrimp, and the right figure is a diagram of the antibacterial effect of the magnolol inclusion compound preparation on intestinal Vibrio of white shrimp. DETAILED DESCRIPTION

[0038] The present invention is described in more detail below through specific implementation modes.

[0039] Example 1

[0040] 1. Extraction and purification of magnolol from Magnolia officinalis by subcritical fluid technology

[0041] The specific extraction and purification method comprises the following steps:

[0042] (1) Pretreatment: Magnolia officinalis bark is crushed into 20 meshes to obtain Magnolia officinalis granular powder, and the Magnolia officinalis powder is put into a 200 mesh silk bag and put into an extraction tank. The equipment is preheated to 60°C, and the vacuum pump is started to pump the pressure in the extraction tank and the separation tank to 0 MPa;

[0043] (2) Extraction: Open the solvent injection valve of the extraction tank, inject the organic solvent propane until the solvent submerges the silk bag and the liquid level is 3 cm above the silk bag, turn on the hot water circulation system pump, maintain the extraction temperature at 30°C and the extraction pressure at 0.2 MPa for 90 minutes to obtain an oil-soluble mixture;

[0044] (3) desolventizing and separating: opening the sample outlet valve of the extraction tank and the sample inlet valve of the separation tank, after the oil-soluble mixture is pressed from the extraction tank into the separation tank, starting the compressor and the condensation system, heating, pressurizing and desolventizing the oil-soluble mixture, and when the pressure of the separation tank drops to 0.10 MPa, starting the vacuum pump and linking with the compressor to accelerate desolventizing and separating, thereby obtaining an organic solvent and a crude extract of Magnolia officinalis.

[0045] (4) Collecting the crude extract of Magnolia officinalis: the organic solvent enters the storage tank for recycling, the discharge valve of the separation tank is opened, the crude magnolol is collected, and the above extraction is repeated 3 times;

[0046] (5) Initial purification of magnolol: the crude extract was dissolved in 8 times the amount of 75% ethanol, heated to 40°C and fully dissolved, then filtered. The filtrate was separated by membrane separation technology, and the components with molecular weight cutoff less than 3000D were ultrafiltered, and then concentrated by nanofiltration membrane with molecular weight less than 200D. After concentration, it was cooled and crystallized to obtain a crude crystalline product;

[0047] (6) Purification of magnolol: The crude crystalline product was dissolved in 2 times the amount of anhydrous ethanol, heated to 40°C until fully dissolved and then filtered. The filtrate was concentrated and cooled to 2°C to crystallize. The purified magnolol was dried at 30°C in vacuo to obtain the magnolol product. The magnolol content was determined by HPLC.

[0048] 2. Preparation of magnolol inclusion complex by saturated solution method

[0049] The specific preparation method comprises the following steps:

[0050] (1) dissolving hydroxypropyl-β-cyclodextrin powder in water, and heating to dissolve to prepare a saturated aqueous solution;

[0051] (2) Dissolve magnolol in 1 volume of 90% ethanol and slowly add it to 5 volumes of saturated solution of hydroxypropyl-β-cyclodextrin under stirring;

[0052] (3) Control the reaction temperature to 15°C, stir the reaction at a stirring speed of 100 r / min for 5 hours, and let it stand for 12 hours;

[0053] (4) Filter out the white crystals, wash the residue once with 5 times 95% ethanol, and dry in an oven at 60°C;

[0054] (5) Moisture content was controlled within 3%, and the mixture was crushed and passed through a 60-mesh sieve to obtain magnolol-β-cyclodextrin inclusion complex.

[0055] 3. Preparation of magnolol inclusion compound preparation

[0056] The specific preparation method is as follows:

[0057] (1) Select appropriate synergists, stabilizers and excipients;

[0058] The synergist is dipotassium hydrogen phosphate; the stabilizer is PEG6000 and EDTA-2 sodium, and the mass ratio of the two is 1:1; the auxiliary material is glucose;

[0059] (2) Screening the optimal addition ratio;

[0060] The mass ratio of the magnolol inclusion compound, the synergist, the stabilizer and the auxiliary materials is 5:2:1:70;

[0061] (3) Prepare into powder, granules, and oral liquid preparations.

[0062] Example 2

[0063] The differences between Example 2 and Example 1 are as follows:

[0064] (1) Extraction and purification of magnolia bark: In the pretreatment step, magnolia bark is crushed to 30 mesh, the preheating temperature is 70°C, and the pressure in the extraction tank and the separation tank is reduced to 0.05 MPa; in the extraction step: the organic solvent is butane, the liquid level is 4 cm higher than the silk bag, the extraction temperature is 55°C, the extraction pressure is 0.5 MPa, and the extraction time is 60 minutes; in the desolventizing and separation step: the pressure in the separation tank is reduced to 0.15 MPa; in the step of collecting the crude extract of magnolia bark: the extraction is repeated 4 times; in the initial purification step of magnolia bark, the crude extract is dissolved in 9 times the amount of 75% ethanol and heated to 45°C to fully dissolve; in the refining step of magnolia bark, the crude crystal product is dissolved in 3 times the amount of anhydrous ethanol, heated to 45°C to fully dissolve, and then filtered, the filtrate is concentrated and cooled to 5°C for crystallization, and vacuum dried at 45°C to obtain a refined magnolia bark product (such as Figure 1 As shown), the purity of magnolol was 98.53% as determined by high performance liquid chromatography (HPLC). Figure 2 shown);

[0065] (2) Preparation of magnolol inclusion complex: magnolol was dissolved in 5 times 80% ethanol, added to 8 times saturated solution of hydroxypropyl-β-cyclodextrin, the reaction temperature was controlled to 25°C, stirred at 300 r / min for 3 hours, allowed to stand for 18 hours, and the filter residue was washed 3 times with 8 times 80% ethanol;

[0066] (3) Preparation of magnolia bark phenol inclusion compound preparation: the synergist is dipotassium hydrogen phosphate and sodium acetate, the mass ratio of the two is 1:1, the stabilizer is β-cyclodextrin, sodium carboxymethyl cellulose, and Tween-80, the mass ratio of the three is 2:1:2; the excipients are glucose and starch, the mass ratio of the two is 1:1; among them, the mass ratio of magnolia bark phenol inclusion compound, synergist, stabilizer and excipients is 10:5:10:75.

[0067] Example 3

[0068] The differences between Example 3 and Example 1 are as follows:

[0069] (1) Extraction and purification of magnolia bark: in the pretreatment step, the bark of magnolia bark is crushed to 50 mesh, the preheating temperature is 75°C, and the pressure in the extraction tank and the separation tank is evacuated to 0.10 MPa; in the extraction step: the organic solvent is liquefied petroleum gas, the liquid level is 5 cm higher than the silk bag, the extraction temperature is 75°C, the extraction pressure is 0.8 MPa, and the extraction time is 30 minutes; in the desolventizing and separation step: the pressure of the separation tank is reduced to 0.20 MPa; in the step of collecting the crude extract of magnolia bark: the extraction is repeated 5 times; in the initial purification step of magnolia bark, the crude extract is dissolved in 10 times the amount of 75% ethanol and heated to 50°C to fully dissolve; in the refining step of magnolia bark, the crude crystal product is dissolved in 5 times the amount of anhydrous ethanol, heated to 50°C to fully dissolve, and then filtered, the filtrate is concentrated and cooled to 8°C for crystallization, and vacuum dried at 60°C to obtain the refined magnolia bark product;

[0070] (2) Preparation of magnolol inclusion complex: magnolol was dissolved in 10 times 70% ethanol, added to 10 times hydroxypropyl-β-cyclodextrin saturated solution, the reaction temperature was controlled to 30°C, stirred at 500 r / min for 1 hour, allowed to stand for 24 hours, and the filter residue was washed 5 times with 10 times 70% ethanol;

[0071] (3) Preparation of magnolia officinalis inclusion compound preparation: disodium hydrogen phosphate and potassium glycyrrhizinate, the mass ratio of the two is 1:1; the stabilizer is β-cyclodextrin and K30, the mass ratio of the two is 2:1; the auxiliary material is starch; among which, the mass ratio of magnolia officinalis inclusion compound, synergist, stabilizer and auxiliary materials is 8:3:6:90.

[0072] Test Example Antibacterial test of magnolol inclusion compound preparation against Vibrio

[0073] Test Example 1 In vitro antibacterial effect test (Vibrio parahaemolyticus)

[0074] 1. Preparation of Vibrio parahaemolyticus liquid:

[0075] (1) The Vibrio parahaemolyticus strains used were purchased from the Institute of Microbiology, Chinese Academy of Sciences;

[0076] (2) Preparation of Vibrio parahaemolyticus liquid: Take Vibrio parahaemolyticus frozen at -80°C, thaw to room temperature, streak on TCBS plate, and culture at 37°C for 24 h;

[0077] (3) Different colonies were selected and placed in TCSB medium, and cultured in a shaking incubator at 37°C and 200 r / min for 24 h to obtain bacterial culture fluid at the initial stage of stability;

[0078] (4) Centrifuge at 4000 r / min for 10 min, resuspend the bacterial pellet in 0.85% sterile saline solution and adjust the concentration to 1 x 106 CFU / mL.

[0079] 2. Preparation of magnolol inclusion compound preparation solution: The magnolol inclusion compound granule preparation prepared in Example 2 of the present invention was dissolved in purified water to form solutions of different concentrations and stored at 4° C. for later use.

[0080] 3. The experimental method and steps for evaluating the effect of magnolol inclusion compound solution on Vibrio parahaemolyticus are as follows:

[0081] (1) Mix the magnolol inclusion compound preparation solution and the bacterial solution in a 1 ml centrifuge tube so that the bacterial solution concentration in the system is about 1 x 106 CFU / ml;

[0082] (2) The concentrations of magnolol inclusion compound preparation solutions were prepared by the doubling dilution method to be 51.2 μg / ml, 25.6 μg / ml, 12.8 μg / ml, 6.4 μg / ml, 3.2 μg / ml, 1.6 μg / ml, 0.8 μg / ml, 0.4 μg / ml, 0.2 μg / ml, and 0.1 μg / ml;

[0083] (3) Incubate the mixture in an oscillating incubator at room temperature and at 100 rpm for 20 min. After the incubation, apply 100 μL of the diluted solution prepared in step 2 to the sample.

[0084] (4) The sample without magnolol treatment was used as the positive control, and the sample without bacterial solution treated with magnolol was used as the negative control;

[0085] (5) Incubate the plate at 37°C for 24 to 48 h and count the number of colonies;

[0086] (6) Three parallel samples were prepared for each treatment, and each dilution was repeated three times;

[0087] (7) The statistical results are shown in the following table. The data were analyzed using analysis software to calculate the minimum inhibitory concentration and minimum bactericidal concentration;

[0088]

[0089] The experimental data show that the minimum bactericidal concentration (MBC) of the magnolol inclusion complex preparation is 25.6 μg / ml, and the minimum inhibitory concentration (MIC) is 12.8 μg / ml.

[0090] Test Example 2 In vivo antibacterial effect test (Penaeus vannamei)

[0091] The experimental methods and steps are as follows:

[0092] 1. Take shrimps from the downwind of the shrimp pond: select 10 white shrimps with full intestines and clear hepatopancreas outlines, peel off the cephalothorax, take out the hepatopancreas and intestines, put them in a clean 10ml beaker, mash them, pour 2ml of clean water into each, stir well, take 0.1ml of tissue fluid from each, drop them into TCBS culture dishes, spread the solution evenly with an L-shaped coating rod, and mark them;

[0093] 2. Place the culture dish in a constant temperature incubator and culture at 37°C for 24 hours. Then take it out and observe and record the number of Vibrio on the culture medium.

[0094] 3. 200 g of the Vibrio inhibiting powder preparation prepared in Example 2 of the present invention was mixed with 40-80 kg of feed, and the mixture was evenly mixed and fed to the whole pond. After 2 hours, the shrimps were collected again, and 10 shrimps with full intestines were selected;

[0095] 4. In the same way as the control group, the hepatopancreas and intestines were dissected and the amount of Vibrio therein was cultured and counted;

[0096] 5. Test results

[0097] The results of hepatopancreas and intestinal antibacterial were as follows: Figure 3 and 4 As shown, Figure 3 The results of the hepatopancreas antibacterial test are as follows: Figure 4 Results of intestinal antibacterial test.

[0098] The in vivo antibacterial test data are shown in the following table:

[0099]

[0100] It can be seen from the data in the above table that the magnolol inclusion compound preparation prepared in Example 2 of the present invention can effectively inhibit Vibrio penelopequine.

[0101] Therefore, the in vitro and in vivo tests on Vibrio (Test Example 1 and Test Example 2) proved that the magnolol inclusion compound preparation of the present invention has a definite inhibitory and killing effect on Vibrio, and the anti-Vibrio effect is significant.

[0102] The above embodiments are merely explanations of the present invention and are not limitations of the present invention. For those skilled in the art, any obvious modifications made thereto without departing from the principles and spirit of the present invention should be considered to be included in the scope of protection of the claims of the present invention.

Claims

1. A magnolol inclusion compound, characterized in that The inclusion compound is prepared from magnolol and hydroxypropyl-β-cyclodextrin, and the specific preparation method comprises the following steps: (1) dissolving hydroxypropyl-β-cyclodextrin powder in water, and heating to dissolve to prepare a saturated aqueous solution; (2) dissolving magnolol with 1 to 10 times of 70 to 90% ethanol, and slowly adding it to 5 to 10 times of saturated solution of hydroxypropyl-β-cyclodextrin under stirring; (3) controlling the reaction temperature to 15 to 30° C., stirring the reaction at a stirring speed of 100 to 500 r / min for 1 to 5 hours, and standing for 12 to 24 hours; (4) Filter out the white crystals, wash the residue 1 to 5 times with 5 to 10 times 70 to 95% ethanol, and dry in an oven at 60°C; (5) Moisture content was controlled within 3%, and the mixture was crushed and passed through a 60-mesh sieve to obtain magnolol-β-cyclodextrin inclusion complex.

2. The magnolol inclusion compound according to claim 1, wherein The magnolol is extracted and purified from Magnolia officinalis, and the specific extraction and purification method comprises the following steps: (1) Pretreatment: Magnolia officinalis bark is crushed into 20-50 meshes to obtain Magnolia officinalis granular powder, the Magnolia officinalis powder is put into a 200-mesh silk bag, and put into an extraction tank, the equipment is preheated to 60-75°C, and the vacuum pump is started to reduce the pressure in the extraction tank and the separation tank to 0-0.10 MPa; (2) Extraction: Open the solvent injection valve of the extraction tank, inject organic solvent propane, butane or liquefied petroleum gas until the solvent submerges the silk bag and the liquid level is 3 to 5 cm higher than the silk bag, turn on the hot water circulation system pump, maintain the extraction temperature at 30 to 75°C, the extraction pressure at 0.2 to 0.8 MPa, and the extraction time is 30 to 90 minutes to obtain an oil-soluble mixture; (3) desolventizing and separating: opening the sample outlet valve of the extraction tank and the sample inlet valve of the separation tank, after the oil-soluble mixture is pressed from the extraction tank into the separation tank, starting the compressor and the condensation system, heating, pressurizing and desolventizing the oil-soluble mixture, and when the pressure of the separation tank drops to 0.10-0.20 MPa, starting the vacuum pump and linking with the compressor to accelerate desolventizing and separating, thereby obtaining an organic solvent and a crude extract of Magnolia officinalis. (4) Collecting the crude extract of Magnolia officinalis: the organic solvent enters the storage tank for recycling, the discharge valve of the separation tank is opened, the crude magnolol is collected, and the above extraction is repeated 3 to 5 times; (5) Initial purification of magnolol: the crude extract was dissolved in 8-10 times the amount of 75% ethanol, heated to 40-50°C and filtered after being fully dissolved. The filtrate was separated by membrane separation technology, and the components with molecular weight cutoff less than 3000D were ultrafiltered, and then concentrated by nanofiltration membrane with molecular weight less than 200D. After concentration, the crude product was cooled and crystallized to obtain a crystalline crude product. (6) Purification of magnolol: The crude crystalline product is dissolved in 2 to 5 times the amount of anhydrous ethanol, heated to 40 to 50°C until fully dissolved and then filtered. The filtrate is concentrated and cooled to 2 to 8°C to crystallize, and vacuum dried at 30 to 60°C to obtain the refined magnolol. The magnolol content is detected by HPLC.

3. A preparation of magnolol inclusion compound as claimed in claim 2, characterized in that: The preparation is prepared by adding a synergist, a stabilizer and an auxiliary material to a magnolol inclusion compound, and the dosage form of the preparation is a powder, a granule or an oral liquid preparation, wherein the mass ratio of the magnolol inclusion compound, the synergist, the stabilizer and the auxiliary material is 5-10:2-5:1-10:70-90.

4. The preparation of magnolol inclusion compound as claimed in claim 3, characterized in that, The mass ratio of the magnolol inclusion compound, the synergist, the stabilizer and the auxiliary materials is 10:5:10:

75.

5. The preparation of the magnolol inclusion compound as claimed in claim 3 or 4, characterized in that, The synergist is one or more of dipotassium hydrogen phosphate, disodium hydrogen phosphate, sodium acetate, and potassium glycyrrhizinate.

6. The preparation of the magnolol inclusion compound as claimed in claim 3 or 4, characterized in that, The stabilizer is one or more of beta-cyclodextrin, sodium carboxymethyl cellulose, PEG6000, K30, sodium EDTA-2, and Tween-80.

7. The preparation of the magnolol inclusion compound as claimed in claim 3 or 4, characterized in that, The auxiliary material is one or more of glucose, starch and anticaking agent.

8. Use of the magnolol inclusion compound preparation as claimed in claim 7 in inhibiting Vibrio in aquaculture.

9. Use of the magnolol inclusion compound preparation as claimed in claim 8 in inhibiting Vibrio in aquaculture, characterized in that: The magnolol inclusion compound preparation can effectively inhibit and kill Vibrio parahaemolyticus in vitro, with a minimum bactericidal concentration MBC of 25.6 μg / ml and a minimum inhibitory concentration MIC of 12.8 μg / ml.

10. Use of the magnolol inclusion compound preparation as claimed in claim 8 in inhibiting Vibrio in aquaculture, characterized in that: The magnolol inclusion compound preparation can effectively inhibit and kill Vibrio in vivo, and 200g of the preparation mixed with 40-80kg of feed can effectively eliminate Vibrio in the intestine and hepatopancreas of white shrimp.

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