CN117122624A - Novel broad-spectrum antibacterial fishing Sha Penghuang ketone preparation and preparation method thereof - Google Patents
Novel broad-spectrum antibacterial fishing Sha Penghuang ketone preparation and preparation method thereof Download PDFInfo
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23K20/00—Accessory food factors for animal feeding-stuffs
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- A23K20/116—Heterocyclic compounds
- A23K20/121—Heterocyclic compounds containing oxygen or sulfur as hetero atom
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
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- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K2236/50—Methods involving additional extraction steps
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Abstract
The invention discloses a preparation method of a Sha Penghuang ketone preparation with low cost and strong practicability, which is characterized in that the preparation method is used for optimizing Sha Penghuang ketone extraction and purification processes, sha Penghuang ketone with the purity of more than or equal to 85 percent can be prepared, the preparation method is characterized in that the preparation method is used for measuring the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) of the prepared Sha Penghuang ketone preparation on the streptococcus tilapiae, the aeromonas hydrophila and the aeromonas verrucosa by taking the streptococcus agalactiae Streptococcus agalactiae (gram positive bacteria, G+), the aeromonas hydrophila Aeromonas hydrophila (gram negative bacteria, G-) and the aeromonas verrucosa Aeromonas veronii (gram negative bacteria, G-) of the aquaculture multiple common tilapia and the aeromonas hydrophila and the aeromonas verrucosa as objects, and the prepared Sha Penghuang ketone preparation has obvious inhibitory effect on the aeromonas hydrophila and has good inhibitory effect on the aeromonas hydrophila and can be used as an antibacterial drug for preventing and controlling diseases for fish.
Description
Technical Field
The invention relates to the field of aquaculture, in particular to a novel broad-spectrum antibacterial fishing Sha Penghuang ketone preparation and a preparation method thereof.
Background
The sand cover (Agriophyllum squarrosum, also called sand rice) is a medical and edible desertification plant, has rich wild resources, is an excellent cattle and sheep feeding plant since ancient times, is used as a traditional Mongolian medicine, has wide and folk clinical application in the northwest, north China and northeast desertification areas of China, is considered to have the functions of clearing heat, detoxicating and dispelling epidemic by Mongolian medicine, and can be used for treating diseases such as epidemic disease, headache, barks, jaundice, kidney heat, urethra burning pain, stomach Heryi, diabetes, aphtha of the tongue, gum ulceration and the like by using sand cover decoction powder or pill powder in the Mongolian medicine division of inner Mongolian medicine and the Chinese medical science, so that the treatment effect of the sand cover on inflammatory reaction is proved to be tested for a long time by clinical practice of Mongolian medicine. However, the application of the samsung in the traditional Chinese medicine is limited to the Mongolian medicine and the folk local application, and the good antibacterial and anti-inflammatory properties of the samsung cannot be paid attention to and developed and utilized in a targeted manner.
The high-density and intensive culture mode in the current aquaculture field has higher and higher pressure on the culture ecological environment, the use of antibiotics becomes the most direct and effective means for ensuring the success of culture by practitioners, the conditions of abuse and disordered use of antibiotics are commonly existed, the problem of drug residue exceeding and detection of disabled drugs and other aquatic animal food safety problems are frequently reported, the 'no-resistance culture' preparation for reducing or even preventing the use of antibiotics is promoted, and some natural medicinal plants capable of playing the roles of resisting bacteria and diminishing inflammation are gradually valued by aquaculture practitioners. However, the price of the common antibacterial anti-inflammatory Chinese herbal medicine raw materials is continuously increased under the influence of the anti-forbidden policy of the livestock breeding industry, and the prepared Chinese patent medicine is mainly simply crushed, so that the effective components are difficult to absorb, the treatment effect is poor, and the traditional Chinese patent medicine is difficult to compare with the common antibiotics. In recent 10 years, the demand of people for natural Chinese herbal medicines is doubled, and Chinese herbal medicine resources are excessively and unordered developed, so that some very common Chinese herbal medicine resources cannot meet the demand.
The Salicornia europaea serving as a natural medicinal plant with good antibacterial and anti-inflammatory effects has extremely large amount of wild resources, and although the application of the Salicornia europaea to aquatic animals is not a precedent, the contained active substance Sha Penghuang ketone has good inhibition effects on common pathogenic bacteria such as water vapor monad, streptococcus agalactiae, aeromonas veronii and the like of the aquatic products, and Sha Penggan medicinal materials are deeply processed, extracted and separated Sha Penghuang ketone is used for treating bacterial diseases of the aquatic animals, so that the promotion of the aquatic product non-chemical cultivation can be effectively promoted, and the food safety of the aquatic products is effectively ensured. Meanwhile, development and utilization of the sand cover are also beneficial to promoting people in the sand area to earn and enrich, so that good social benefit is brought, and win-win is achieved.
Disclosure of Invention
The invention aims to seek novel natural medicines to treat common bacterial diseases of aquaculture and reduce the use of antibiotics in the aquaculture process, thereby disclosing the novel broad-spectrum antibacterial natural Mongolian medicine plant Salicornia europaea for fishing and the function of antibacterial active ingredient Salicornia europaea flavone thereof in coping with common pathogenic bacteria of aquaculture, and disclosing a preparation method with low preparation cost and strong operability.
In the invention, the Sha Penghuang ketone preparation is prepared by extraction through the following ways: collecting dried plant of Salicornia Herbacea of wild annual medicinal plant at the edge of Tenggrei desert, removing the whole plant of seed ear, crushing into Salicornia Herbacea micropowder capable of passing through 200 mesh sieve, ultrasonic extracting with 95% ethanol, filtering to obtain ethanol extractive solution, recovering ethanol under reduced pressure to obtain pasty ethanol extract, adding 5L pure water for redissolution, filtering to remove water insoluble substances, collecting filtrate, concentrating under reduced pressure to solid state, pulverizing, and sieving with 200 mesh sieve to obtain Sha Penghuang ketone preparation.
The Sha Penghuang ketone is orally taken in an amount of 0.5-2g of Sha Penghuang ketone preparation per kilogram of fish body weight per day. The oral feeding method comprises the steps of accurately weighing the Sha Penghuang ketone preparation required after the dosage of the Sha Penghuang ketone preparation is calculated according to the weight of the fish, dissolving the Sha Penghuang ketone preparation in a proper amount of clean water, uniformly mixing all water-soluble substances into the feed, and drying the feed mixed with the Sha Penghuang ketone preparation in the shade for 1h to feed the feed. The mixture is fed with Sha Penghuang ketone preparation 1 meal every day, and the mixture is continuously fed for 7 days.
According to the invention, the antibacterial test by an oxford cup method and a turbidimetry proves that the multiple ratio diluent of the Sha Penghuang ketone preparation has a certain inhibition effect on aeromonas hydrophila Aeromonas hydrophila, aeromonas veronii Aeromonas veronii, tilapia streptococcus agalactiae Streptococcus agalactiae, pseudomonas aeruginosa Pseudomonas aeruginosa, serratia marcescens Serratia marcescens, hafnia alvei, stenotrophomonas maltophilia Stenotrophomonas maltophilia and pseudomonas fluorescens Pseudomonas fluorescens, and the prepared Sha Penghuang ketone preparation has the characteristic of broad antibacterial spectrum. Meanwhile, the prepared Sha Penghuang ketone preparation has the strongest antibacterial effect on aeromonas hydrophila and also has stronger antibacterial effect on aeromonas verrucosa and tilapia streptococcus agalactiae, which indicates that the prepared Sha Penghuang ketone preparation can be used as an antibacterial drug for preventing and controlling bacterial diseases of aquaculture.
Compared with the existing fishing antibiotic substitute and technology, the invention has the following advantages: 1. the novel broad-spectrum antibacterial fishing Sha Penghuang ketone preparation is prepared by extracting dried plants of wild annual plant suaeda salsa with extremely large biomass at the edge of a Tenggre desert, and has the advantages of easily available raw materials, lower cost and easy popularization; 2. according to the invention, sha Penghuang ketone is used for preventing and controlling bacterial diseases of aquaculture for the first time, so that the antibacterial effect is good, the antibacterial spectrum is wide, and the development of the non-resistance aquaculture is promoted; 3. the preparation method adopted by the invention not only completes the deep processing of the raw materials of the samsung, but also avoids the problems of high cost and difficult popularization and application of the conventional fine deep processing technology, and directly prepares the Sha Penghuang ketone preparation with high content, solves the problems of long treatment course, slow effect, poor effect, low utilization rate and high use cost of the common Chinese herbal medicine preparation, avoids the waste of natural medicinal plant resources, reduces the medication cost for the breeding practitioner and reduces the disease loss caused by improper medication; 4. the method can be used for developing and utilizing the sand-flea resources in northwest, north China and northeast remote areas, can also increase income sources for local poor population, and has higher social benefit.
Detailed Description
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are provided only for illustrating the present invention and are not to be construed as limiting the scope of the present invention
Example 1: determination of minimum inhibitory concentration and minimum bactericidal concentration of the Sha Penghuang ketone formulation on aeromonas hydrophila (G-).
1g of Sha Penghuang ketone preparation is weighed and dissolved in 10ml of deionized water, filtered and sterilized by a filter membrane with the aperture of 0.22 um, and diluted by a sterile PBS buffer solution for later use. Aeromonas hydrophila is gram negative bacteria (G-), and is derived from aquatic animal pathogen library of Shanghai ocean university country, and the strain number is 1019. Inoculating the strain into sterile LB culture solution, activating and culturing overnight at 30deg.C, and suspending the obtained strain until OD600 value is 0.1-0.3. 100ul of LB culture solution, 100ul of aeromonas hydrophila solution and 50ul of Sha Penghuang ketone preparation multiple ratio diluent with different concentrations are sequentially added into a sterile 96-well plate, and after 18 h of culture at 30 ℃, the OD600 value of each well is measured by an enzyme-labeled instrument. The control group used an equivalent amount of sterilized PBS buffer instead of Sha Penghuang ketone formulation-fold dilution. The minimum concentration of Sha Penghuang ketone formulation that the OD600 value no longer increased compared to the initial value was Sha Penghuang ketone formulation Minimum Inhibitory Concentration (MIC) against aeromonas hydrophila. Sequentially taking the mixed solution from the 96-well plate, inoculating the mixed solution to a solid LB culture medium plate, culturing at 30 ℃ for 24 h, observing the formation of aseptic colonies, and obtaining the Sha Penghuang ketone preparation concentration corresponding to the solid LB culture medium plate formed by aseptic colonies, namely the minimum sterilization concentration (MBC) of the Sha Penghuang ketone preparation on aeromonas hydrophila.
Example 2: determination of minimum inhibitory concentration and minimum bactericidal concentration of aeromonas veronii (G-) by Sha Penghuang ketone formulation.
1g of Sha Penghuang ketone preparation is weighed and dissolved in 10ml of deionized water, filtered and sterilized by a filter membrane with the aperture of 0.22 um, and diluted by a sterile PBS buffer solution for later use. Aeromonas veronii is gram-negative bacteria (G-), and is derived from aquatic animal pathogen library of Shanghai ocean university country, and the strain number is 1024. Inoculating the strain into sterile LB culture solution, activating and culturing overnight at 30deg.C, and suspending the obtained strain until OD600 value is 0.1-0.3. 100ul of LB culture solution, 100ul of Aeromonas veronii solution and 50ul of Sha Penghuang ketone preparation multiple ratio dilution with different concentrations are sequentially added into a sterile 96-well plate, and after 18 h of culture at 30 ℃, OD600 values of all the wells are measured by an enzyme-labeled instrument. The control group used an equivalent amount of sterilized PBS buffer instead of Sha Penghuang ketone formulation-fold dilution. The minimum concentration of Sha Penghuang ketone formulation that the OD600 value no longer increased compared to the initial value was Sha Penghuang ketone formulation Minimum Inhibitory Concentration (MIC) against aeromonas veronii. Sequentially taking the mixed solution from the 96-well plate, inoculating the mixed solution to a solid LB culture medium plate, culturing at 30 ℃ for 24 h, observing the formation of aseptic colonies, and obtaining the Sha Penghuang ketone preparation concentration corresponding to the solid LB culture medium plate formed by aseptic colonies, namely the minimum sterilizing concentration (MBC) of Sha Penghuang ketone on aeromonas veronii.
Example 3: determination of minimum inhibitory concentration and minimum bactericidal concentration of the Sha Penghuang ketone formulation on tilapia streptococcus agalactiae (g+).
1g of Sha Penghuang ketone preparation is weighed and dissolved in 10ml of deionized water, filtered and sterilized by a filter membrane with the aperture of 0.22 um, and diluted by a sterile PBS buffer solution for later use. The streptococcus agalactiae of tilapia is gram positive bacteria (G+), and is derived from aquatic animal pathogen library of Shanghai ocean university country, and the strain number is. Inoculating the strain into a sterile sheep blood culture solution, and performing activation culture at 30deg.C overnight to obtain strain with OD600 value of 0.1-0.3. 100ul of sheep blood culture solution, 100ul of tilapia streptococcus agalactiae bacteria solution and 50ul of Sha Penghuang ketone preparation multiple ratio diluent with different concentrations are sequentially added into a sterile 96-well plate, and after culturing for 18 h at 30 ℃, the OD600 value of each well is measured by an enzyme-labeling instrument. The control group used an equivalent amount of sterilized PBS buffer instead of Sha Penghuang ketone formulation-fold dilution. The minimum concentration of Sha Penghuang ketone formulation that the OD600 no longer increases compared to the initial value is Sha Penghuang ketone formulation's Minimum Inhibitory Concentration (MIC) against tilapia streptococcus agalactiae. Sequentially taking the mixed solution from the 96-well plate, inoculating the mixed solution to a sheep blood culture medium plate, culturing at 30 ℃ for 24 h, observing the formation of aseptic colonies, and obtaining the Sha Penghuang ketone preparation concentration corresponding to the sheep blood culture medium plate formed by aseptic colonies, namely the minimum sterilization concentration (MBC) of the Sha Penghuang ketone preparation on the tilapia streptococcus agalactiae.
Claims (6)
1. A novel broad-spectrum antibacterial fishing preparation contains Sha Penghuang ketone as main ingredient.
2. The method for preparing the novel broad-spectrum antibacterial fishing Sha Penghuang ketone preparation according to claim 1, wherein the method is characterized in that ethanol is added for ultrasonic extraction after the sand is crushed, residues are removed by filtration to obtain a crude extract, the crude extract is concentrated by recovering ethanol under reduced pressure and then is completely dissolved in water, water insoluble substances are filtered out, the filtrate is concentrated to be solid under reduced pressure, and the Sha Penghuang ketone preparation is obtained after crushing.
3. The method for preparing the novel broad-spectrum antibacterial fishing Sha Penghuang ketone preparation according to claim 1, wherein dried plants of wild annual medicinal plants, such as Salicornia herbacea, at the edge of a Tenggrei desert are collected, the whole plants are sheared and crushed into particles with the particle size of 5-10mm after seed ears are removed, and the particles are sent to a closed pulverizer to be crushed, so that Salicornia herbacea micropowder with the particle size of about 100 mu m and capable of being sieved by a 200-mesh sieve is prepared as an extraction raw material.
4. The preparation method of the novel broad-spectrum antibacterial fishing Sha Penghuang ketone preparation according to claim 1, which is characterized in that 5L of 95% ethanol is added into each kilogram of sand-fleshed micro powder, the ultrasonic wall is broken, the ultrasonic power is 800W, the ultrasonic time is 10min, after 2 times of continuous ultrasonic treatment, the ethanol extract is filtered, the ethanol is recovered under reduced pressure to obtain a pasty ethanol extract, 5L of pure water is added for re-dissolution, water insoluble substances are filtered, the filtrate is collected, the filtrate is concentrated to be solid under reduced pressure, and the filtrate is crushed and filtered through a 200-mesh sieve to obtain the Sha Penghuang ketone preparation.
5. A novel broad spectrum antibacterial formulation of fishing Sha Penghuang ketone, according to claim 1, for oral administration in an amount of from 0.5g to 2g per kg of body weight of fish per day of Sha Penghuang ketone formulation per kg of body weight of fish.
6. According to claim 5, a novel broad-spectrum antibacterial fishing Sha Penghuang ketone preparation is orally fed by accurately weighing the required Sha Penghuang ketone preparation after the Sha Penghuang ketone preparation dosage is calculated according to the fish weight, dissolving the required Sha Penghuang ketone preparation in a proper amount of clean water, uniformly mixing all water-soluble substances into feed, and drying the feed mixed with the Sha Penghuang ketone preparation in the shade for 1 h. The mixture is fed with Sha Penghuang ketone preparation 1 meal every day, and the mixture is continuously fed for 7 days.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202210537580.2A CN117122624A (en) | 2022-05-18 | 2022-05-18 | Novel broad-spectrum antibacterial fishing Sha Penghuang ketone preparation and preparation method thereof |
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| CN202210537580.2A CN117122624A (en) | 2022-05-18 | 2022-05-18 | Novel broad-spectrum antibacterial fishing Sha Penghuang ketone preparation and preparation method thereof |
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| CN117122624A true CN117122624A (en) | 2023-11-28 |
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