CN115944659B - Mangosteen extract and application of mangosteen extract as active ingredient - Google Patents

Mangosteen extract and application of mangosteen extract as active ingredient Download PDF

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CN115944659B
CN115944659B CN202310080173.8A CN202310080173A CN115944659B CN 115944659 B CN115944659 B CN 115944659B CN 202310080173 A CN202310080173 A CN 202310080173A CN 115944659 B CN115944659 B CN 115944659B
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mangostin
nocardia
alpha
mangosteen
mangosteen extract
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CN115944659A (en
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王文博
焦铁军
金湛林
张中雨
卢静
马瑞
孟强
蔡畅敏
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Shenzhen Wankesen Biotechnology Co ltd
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Abstract

The invention belongs to the technical field of aquaculture, relates to prevention and treatment of nocardia, and in particular relates to application of mangosteen extract and active ingredients thereof in preparation of nocardia prevention and treatment medicines. The invention provides a technical idea of using Chinese herbal medicine extracts and active ingredients thereof for preparing medicines or feed additives for preventing and treating nocardia, and discloses the inhibition activity of mangosteen extracts and active ingredients thereof on nocardia for the first time, wherein the minimum inhibitory concentration of alpha-mangostin on nocardia is 3.125-6.25 mug/mL, which is lower than the minimum inhibitory concentration of enrofloxacin hydrochloride, ciprofloxacin hydrochloride, norfloxacin, florfenicol, sulfamethoxazole, neomycin and other aquatic antibiotics. This suggests that α -mangostin has the potential to be directly developed as an anti-nocardia drug.

Description

Mangosteen extract and application of mangosteen extract as active ingredient
Technical Field
The invention belongs to the technical field of aquaculture, relates to prevention and treatment of nocardia, and in particular relates to application of mangosteen extract and active ingredients thereof in preparation of nocardia prevention and treatment medicines.
Background
Nocardia is a gram-positive pathogen belonging to the genus nocardia, phylum actinomycetes, order actinomycetes, family nocardiaceae, genus nocardia, and is a variety of nocardia species, which are widely present in soil, dust, sea fresh water, rotten vegetation and animal excretions. Nocardia in fish is a common chronic systemic granulomatous disease caused by nocardia, and is extremely harmful to aquaculture and safety of aquatic products. At present, nocardia infecting fishes in aquaculture is mainly nocardia seriiolae @Nocardia seriolae) Can cause the infection of more than 20 important economic fishes such as snakehead, larch, tilapia, and sea water cultured Seriola, porgy, pomfret, grouper, large yellow croaker, and the like. With the continuous development of water plant breeding industry in recent years, the continuous improvement of the intensive degree of breeding and the continuous increase of the communication of fish seedlings in various places, the transmission range of nocardia becomes wider, the morbidity is higher, the hazard degree is deeper, and huge economic loss is caused for the water production breeding industry.
Mangosteen (mangosteen)Garcinia mangostanaL.) is fruit of Garcinia genus (Guttirae) of Guttirae, which is widely distributed in southeast Asia countries such as Malaysia, myma, thailand, and has been cultivated in large quantities. The mangosteen is rich in flavone, polysaccharide, protein, lipid and other compounds, and is a traditional medicinal plant commonly used in Dai nationality and southeast Asian countries. The mangosteen fruit mainly comprises xanthone compound. Modern pharmacology shows that the mangosteen fruit has various biological activities such as anti-inflammatory, antibacterial, antioxidant and the likeOxa-allinones present in the pericarp have inhibitory effect on Fatty Acid Synthase (FAS) full response. 3 of mangosteen (7-week maturation period) contain prenyl groups that are resistant to human cancer cells. The mangosteen can accelerate the healing of skin wound surfaces of guinea pigs and rejuvenate wound surfaces.
Nocardia in fish is a chronic granulomatous disease. Fish will show a slow response after infection, a marked decrease in swimming power, sinking to the water, and a decrease in appetite. With the exacerbation of the disease, the liver, spleen, kidneys, etc. have white nodules with diameters of 1-5 mm. The nocardia of fish has the characteristics of high infection rate, high death rate, long incubation period and long disease period, has no obvious characteristics at the initial stage of disease, and is difficult to detect. Usually, when the cultured fish grows into adult fish, the body surface and viscera tissues of the cultured fish are seriously damaged, and the commodity value of the adult fish is seriously affected. In addition, nocardia in fish is also a facultative intracellular pathogen, which can resist the sterilization activities of neutrophils, monocytes and macrophages, which presents great difficulty in the treatment of nocardia in fish. At present, no optimal preventive medicine is established for fish nocardia, and methods such as timed pond searching, preventive management and disinfection of culture water body by non-oxidative bactericides are generally adopted. At present, no optimal therapeutic medicine is established for fish nocardia, the development of vaccines is slow, and the medicine treatment mainly depends on trimethoprim-disulfonate and antibiotics. At present, the trimethoprim-disulfo tablet has great danger to people, is strictly forbidden to be used for food and feed processing, and the phenomenon of drug resistance generally occurs when antibiotics are excessively used. Therefore, there is an urgent need to develop a safe, effective and environmentally friendly method for preventing and treating nocardia in fish.
Disclosure of Invention
The invention aims to solve the problem that antibiotics are commonly adopted in the treatment process of fish nocardia, and the excessive use of the antibiotics causes drug resistance; the fish nocardia disease is difficult to prevent, and has no obvious characteristics at the initial stage of the disease.
Based on the above objects, the present application solves such technical problems in the art by providing an application of mangosteen extract and active ingredients thereof.
In one aspect, the invention relates to the use of alpha-mangostin or a pharmaceutically acceptable salt thereof in the preparation of a nocardia control drug.
In another aspect, the invention relates to the use of alpha-mangostin or a pharmaceutically acceptable salt thereof in the preparation of a feed additive for the control of nocardia.
In another aspect, the invention relates to the use of mangosteen extract in the preparation of nocardia control drugs.
In another aspect, the invention relates to the use of mangosteen extract in the preparation of a feed additive for the control of nocardia.
The invention is not particularly limited to the preparation method of the mangosteen extract, and generally, three methods of preparing the mangosteen extract are provided, namely a heating ultrasonic reflux extraction method, a methanol cold leaching method and a critical extraction method. The mangosteen extract comprises at least one of alpha-mangostin, beta-mangostin, gamma-mangostin and iso-mangostin as an active ingredient.
Illustratively, the present invention provides a method for preparing a mangosteen extract by heating ultrasonic reflux extraction, comprising: drying mangosteen pericarp in a constant temperature oven at 55 ℃ to constant weight, crushing the mangosteen pericarp by using a miniature plant sample crusher, and sieving the mangosteen pericarp with a 40-60-mesh sieve; extracting crushed and sieved mangosteen pericarp with methanol twice with solvent of 250mL each time and reflux time of 3h, performing ultrasonic treatment for 5min every 30 min, filtering the extractive solution after each extraction, concentrating under reduced pressure and heating with a rotary evaporator, mixing the concentrated extracts for 2 times, and oven drying at 55deg.C to constant weight to obtain mangosteen extract.
Illustratively, the present invention provides a method for preparing mangosteen extract by a methanol cold-dip process, comprising: drying mangosteen pericarp in a constant temperature oven at 55 ℃ to constant weight, crushing the mangosteen pericarp by using a miniature plant sample crusher, and sieving the mangosteen pericarp with a 40-60-mesh sieve; every 100g of mangosteen pericarp after crushing and sieving is added with 1500mL of methanol, cold soaking is carried out for 24h, and after suction filtration, the mangosteen extract is obtained by decompression, heating and concentration by a rotary evaporator.
Illustratively, the present invention provides supercritical CO 2 A method for preparing a mangosteen extract by extraction, comprising: drying mangosteen pericarp in constant temperature oven at 55deg.C to constant weight, pulverizing with miniature plant sample pulverizer, pulverizing mangosteen pericarp, and placing into extraction kettle, and adding CO 2 And (3) respectively heating the extraction kettle, the analysis kettle I and the analysis kettle II to obtain an extractant, pressurizing the extraction kettle and the two analysis kettles by a high-pressure pump when the temperature reaches 35 ℃ and 40 ℃ and 37 ℃ respectively, and starting to circularly extract and keeping constant temperature and constant pressure when the pressure reaches 22mPa, 7.5mPa and 6mPa respectively, wherein the extraction time is 4h, and discharging from the analysis kettle I and the analysis kettle II every 10min to obtain the mangosteen extract.
The invention does not particularly limit the preparation method of alpha-mangostin, beta-mangostin, gamma-mangostin or iso-mangostin, and generally, the alpha-mangostin, beta-mangostin, gamma-mangostin or iso-mangostin can be obtained from mangosteen extract.
In another aspect, the invention relates to a medicament for controlling nocardia comprising α -mangostin.
In another aspect, the invention relates to a feed additive for controlling nocardia comprising α -mangostin.
In another aspect, the invention relates to a medicament for controlling nocardia comprising mangosteen extract.
In another aspect, the invention relates to a feed additive for controlling nocardia comprising mangosteen extract.
The term "control" as used herein means preventing or reducing nocardia enrichment, curing or reducing nocardia after use in the presence of possible nocardia or factors that cause nocardia.
In the present invention, the term "pharmaceutically acceptable" means that there is no long-term detrimental effect on the general health of the subject being treated.
In the present invention, the term "pharmaceutically acceptable salt" refers to a salt that retains the biological efficacy of α -mangostin, β -mangostin, γ -mangostin, or iso-mangostin, and has no adverse effect in biology or other aspects. Pharmaceutically acceptable salts refer to inorganic or organic acid salts which convert the base group of the parent compound to a salt form, such as a base group (e.g., amine group), and are typically prepared by reacting the parent compound with a conventional type of acid in a solvent system, and inorganic acids typically include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; the organic acid generally comprises acetic acid, propionic acid, glycollic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.
Compared with the prior art, the invention has the following beneficial effects or advantages:
(1) The mangosteen extract and the active ingredients thereof, especially alpha-mangostin, are used as a natural compound derived from Chinese herbal medicines, and have the advantages of environmental friendliness, low drug resistance, less environmental residue, low biotoxicity, natural and easy degradation and the like. The mangosteen extract and the active ingredients thereof are used for preparing medicines for preventing and treating nocardia or feed additives for preventing and treating nocardia, can effectively relieve the problems of medicine residue, environmental pollution and drug resistance caused by chemical medicines and antibiotics, and completely meet the requirements of the current green pollution-free aquatic products.
(2) According to the invention, the minimum inhibitory concentration (Minimum inhibitory concentration, MIC) of the alpha-mangostin, the beta-mangostin, the gamma-mangostin, the iso-mangostin and other bioactive components on nocardia is measured according to a standard microdilution method recommended by national standards committee (NCCLS) of clinical laboratories, experiments show that the MIC values of the mangosteen extract and the beta-mangostin are 25-50 mug/mL, the MIC value of the iso-mangostin is 25-100 mug/mL, the MIC value of the gamma-mangostin is 12.5-25 mug/mL, the MIC value of the alpha-mangostin is 3.125-6.25 mug/mL, and the mangosteen extract and the activity thereof all show excellent antibacterial performance. Wherein, the MIC value of the alpha-mangostin is lower than that of the aquatic antibiotics approved by agricultural parts such as enrofloxacin hydrochloride, ciprofloxacin hydrochloride, norfloxacin, florfenicol, sulfamethazine, neomycin and the like, which shows that the mangosteen extract and the activity thereof, in particular the alpha-mangostin has the capability of being directly developed into the nocardia resistant drug.
(2) The invention uses alpha-mangostin for preparing nocardia prevention and treatment medicaments, and prepares injection, which can obviously reduce the copy number (P < 0.01) of nocardia in the liver of the largehead jewfish, wherein the copy number reduction multiples of nocardia in the treatment groups of 20mg/kg, 40mg/kg and 80mg/kg are 5.24 times, 9.67 times and 8.13 times respectively.
(3) The alpha-mangostin is used for preparing the feed additive for preventing and treating nocardia, and the prepared feed additive has excellent treatment effect on nocardia at the additive amount of 200mg/kg, 400mg/kg and 800mg/kg, wherein the additive amount of 400mg/kg has the best treatment effect, the survival rate of the nocardia infected larceny jetsoever is improved to 78.6%, and the inhibition effect on proliferation of nocardia in fish bodies is the best.
(4) According to the invention, the alpha-mangostin is used for preparing the nocardia control drug, and is prepared into an injection, when the injection dose of the alpha-mangostin is 40mg/kg and the alpha-mangostin is used for preparing the nocardia control drug, the infection rate of the largehead jewfish is 0, the survival rate reaches 100%, and the preparation of the alpha-mangostin used for preparing the nocardia control drug is illustrated, so that the nocardia infection in a natural state can be effectively prevented, and the occurrence of the nocardia in fish can be effectively prevented.
Drawings
FIG. 1 is a chromatogram of the alpha-mangostin standard HLPC.
FIG. 2 is a HLPC chromatogram of a mangosteen extract sample.
FIG. 3 shows nocardia content of liver of Lateolabrax megacephalae 4 days after nocardia infection.
FIG. 4 shows nocardia content of liver of Lateolabrax megacephalae 7 days after nocardia infection.
Fig. 5 shows survival rate of micropterus salmoides after 21 days of detoxification.
Description of the embodiments
The following describes the technical aspects of the present invention with reference to examples, but the present invention is not limited to the following examples.
In order that those skilled in the art will better understand the technical solution of the present invention, the present invention will be further described with reference to the specific examples and the accompanying drawings, but the examples are not intended to be limiting.
The experimental methods and the detection methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.
Examples
The present example provides the preparation and identification of alpha-mangostin and mangosteen extracts.
Preparation of mangosteen extract (supercritical CO 2 Extraction method): the mangosteen is treatedGarcinia mangostanaL.) the pericarp is dried to constant weight in a constant temperature oven at 55 ℃, and after being dried, the pericarp is crushed by a miniature plant sample crusher and then is put into an extraction kettle. Heating the extraction kettle, the analysis kettle I and the analysis kettle II respectively, pressurizing the extraction kettle and the two analysis kettles by a high-pressure pump when the temperature reaches 35 ℃ and 40 ℃ and 37 ℃ respectively, and starting to circularly extract and keeping constant temperature and constant pressure when the pressure reaches 22mPa, 7.5mPa and 6mPa respectively, wherein the extraction agent is carbon dioxide, the extraction time is 4h, and discharging from the analysis kettle I and the analysis kettle II every 10min, thereby obtaining the mangosteen extract.
The identification method of alpha-mangostin, beta-mangostin, gamma-mangostin and iso-mangostin comprises the following steps: precisely weighing 1mg of mangosteen extract, dissolving with methanol, fixing volume to 25mL, and filtering with 0.45 μm filter membrane to obtain sample preparation solution; precisely weighing an alpha-mangostin standard substance, dissolving with methanol to constant volume, and respectively preparing into 200, 100, 50, 25, 12.5, 6.25 and 3.125 mug/mL standard solutions; 10. Mu.L of the sample was injected into a liquid chromatograph-mass spectrometer (LC-MS) for measurement. The mobile phase is acetonitrile-water (85:15), the liquid chromatographic column is Waters symmetry C-18 (4.6 mm. Times.150 nm), the volume flow is 1mL/min, the detection wavelength is 280nm, the column temperature is 30 ℃, and a standard curve is drawn. And then carrying out liquid phase-mass spectrometry on the sample liquid.
Those skilled in the art will readily appreciate that mangosteen extract contains α -mangostin, β -mangostin, γ -mangostin, and iso-mangostin. In this example, alpha-mangostin was used as an example, and the identification of the alpha-mangostin and mangosteen extract was shown in FIGS. 1 and 2. FIG. 1 is a chromatogram of the alpha-mangostin standard HLPC. FIG. 2 is a HLPC chromatogram of a mangosteen extract sample.
The time at which a peak occurs in the liquid chromatogram is referred to as the retention time or retention value. Under certain chromatographic conditions, an unknown has only a certain retention time. Thus, the retention time of a known pure substance under the same chromatographic conditions can be compared with the retention time of an unknown substance to qualitatively identify the unknown substance. The method for determining alpha-mangostin in this embodiment is a method for qualitative analysis by liquid chromatography, which comprises drawing a standard curve with alpha-mangostin standard substance, and determining retention time. Subjecting the mangosteen extract to liquid chromatography under the same chromatographic conditions, and comparing the retention time of the obtained liquid chromatogram with that of a standard curve.
As can be seen from fig. 1 and fig. 2, the peak values of the retention time in fig. 2, in which the retention time of the third distinct peak is the same as that of the peak value in the HLPC chromatogram of the α -mangostin standard product in fig. 1, indicate that the obtained mangosteen extract contains α -mangostin.
Examples
This example provides the determination of in vitro antimicrobial MIC values for α -mangostin, β -mangostin, γ -mangostin, iso-mangostin.
In this example, 8 nocardia strains isolated from fish with nocardia in a farm were used, and the strain numbers are shown in table 1. The Minimum Inhibitory Concentration (MIC) of bioactive components of alpha-mangostin, beta-mangostin, gamma-mangostin and iso-mangostin in mangosteen extract on 8 nocardia strains is determined, and various common antibiotics are used as a control.
Through the identification and the purification, the physiological biochemistry, the molecular biology and other conventional technical means, 8 nocardia strains are nocardia seriolaeN. seriolae）。
TABLE 1 Nocardia Seriola strain origin
Strain numbering Bacterial strain origin Separation part
NC61-AKLIL-0502 Shanxi Ankang farm Liver of weever
NC61-AHSPL-0401 Shanxi Hanzhong farm Spleen of weever
NC51-MYLIW-0602 Sichuan Mianyang farm Snakehead liver
NC51-LZSPW-0307 Sichuan Luzhou farm Snakehead spleen
NC51-YBLIL-0403 Sichuan yibine farm Liver of weever
NC44-CZLIS-0305 Guangdong Chaozhou farm Liver of Serissa japonica
NC44-MMSPS-0406 Guangdong MaoMing farm Spleen of Serissa japonica
NC44-MMSPS-0407 Guangdong MaoMing farm Liver of weever
The bacterial (strain) culture solution adopts soybean casein liquid culture medium (TSB). The preparation method of the TSB comprises the following steps: 3.0g of tryptone, 1.0g of soytone and 1.0g of sodium chloride are weighed, 200mL of distilled water is added, the mixture is heated and dissolved, then the mixture is cooled to room temperature, 5mol/L hydrochloric acid is used for adjusting the pH to 7.3, the mixture is autoclaved for 15 minutes at 121 ℃, and the mixture is cooled to the room temperature for standby. Inoculating near the flame of alcohol lamp of super clean bench, and culturing in constant temperature incubator at 28deg.C for 5 days. 5mL of the cultured bacterial liquid is placed in a 10mL centrifuge tube, and the concentration of the bacterial liquid is determined to be 1.0x10 by using a Mahalanobis turbidimetry method 8 cfu/mL, diluted to 1.0X10 with unvaccinated sterilized TSB medium 5 cfu/mL is reserved.
Preparing a medicine mother solution: a certain amount of mangosteen extract (preparation see example 1) was accurately weighed with an analytical balance, the active ingredients of the mangosteen extract (α -mangostin, β -mangostin, γ -mangostin, iso-mangostin), antibiotics (enrofloxacin hydrochloride, levofloxacin hydrochloride, ciprofloxacin hydrochloride, florfenicol, norfloxacin, sulfamonomethoxine and neomycin sulfate) were dissolved in DMSO at a concentration of 20 μg/mL, and the mixture was packed in 1.5mL centrifuge tubes. The mother liquor was passed through a 0.22 μm filter and diluted in sterile TSB medium in multiple ratios for MIC determination.
The Minimum Inhibitory Concentration (MIC) of each of the above drugs was determined according to the standard microdilution method recommended by the national standards institute (NCCLS) of clinical laboratories. Taking 96-well plate, under the aseptic condition of the super clean bench, according to experimental drugsThe number of columns was set, and 100. Mu.L of nocardia liquid (1X 10) was added to each well of each column 5 cfu/mL); 100 mu L of drug solution diluted by a ratio of 100 mu L is sequentially added from the first hole to the 8 th hole of each column, 3 empty holes are selected, 100 mu L of nocardia solution and 100 mu L of sterile TSB solution are added as a control, and 200 mu L of sterile distilled water is added to the rest empty holes for reducing water vapor evaporation. The 96-well plate is covered, and is placed in a constant temperature incubator at 28 ℃ for culture, 50 mu L of blue resazurin solution is added into each well after 5 days, and the observation result is 24 hours later, according to the principle of the resazurin redox reaction, the red-changed well indicates that bacteria grow, and the drug concentration without color change is the minimum concentration MIC of the drug for inhibiting bacteria growth. The MIC values of mangosteen extract, alpha-mangostin, beta-mangostin, gamma-mangostin, iso-mangostin against strain 1 are shown in Table 2.
TABLE 2 antibacterial MIC of mangosteen extract active ingredient (μg/mL)
Strain numbering Mangosteen extract Alpha-mangostin Beta-mangostin Gamma-mangostin Iso-mangostin
NC61-AKLIL-0502 25 6.25 50 25 100
NC61-AHSPL-0401 50 6.25 50 25 100
NC51-MYLIW-0602 25 3.125 25 12.5 50
NC51-LZSPW-0307 25 3.125 25 12.5 50
NC51-YBLIL-0403 25 3.125 25 12.5 50
NC44-CZLIS-0305 50 6.25 50 25 100
NC44-MMSPS-0406 25 3.125 50 25 50
NC44-MMSPS-0407 25 3.125 50 25 50
As is clear from Table 2, mangosteen extract, α -mangostin, β -mangostin, γ -mangostin, iso-mangostin exhibited excellent antibacterial properties against 8 nocardia strains. The antibacterial performance of the alpha-mangostin is particularly outstanding, and the MIC value of the alpha-mangostin to 8 nocardia strains is 3.125-6.25 mug/mL. Therefore, the mangosteen extract and the active ingredients thereof, especially the alpha-mangostin, can be judged, and the potential of directly developing the mangosteen extract into the nocardia resistant medicament is provided.
MIC values of common antibiotics (enrofloxacin hydrochloride, levofloxacin hydrochloride, ciprofloxacin hydrochloride, florfenicol, norfloxacin, sulfamonomethoxine and neomycin sulfate) for 8 nocardia strains are shown in table 3.
TABLE 3 antibacterial MIC of common antibiotics (μg/mL)
Strain numbering Enrofloxacin hydrochloride Levofloxacin hydrochloride Ciprofloxacin hydrochloride Florfenicol Norfloxacin Norfloxacin Sulfadimethoxypyrimidine Sulfamethoxypyrimidine Neomycin sulfate
NC61-AKLIL-0502 25 12.5 25 6.25 25 100 25 50 3.125
NC61-AHSPL-0401 25 12.5 25 6.25 25 100 50 50 3.125
NC51-MYLIW-0602 12.5 25 12.5 12.5 50 100 50 50 3.125
NC51-LZSPW-0307 12.5 25 25 12.5 50 100 25 25 6.25
NC51-YBLIL-0403 12.5 25 25 12.5 50 100 50 50 3.125
NC44-CZLIS-0305 25 12.5 25 6.25 25 100 50 50 6.25
NC44-MMSPS-0406 50 12.5 12.5 12.5 25 100 50 25 3.125
NC44-MMSPS-0407 50 12.5 12.5 12.5 25 100 50 25 3.125
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As can be seen from Table 3, the mangosteen extract, alpha-mangostin, beta-mangostin, gamma-mangostin and iso-mangostin showed better antibacterial activity against 8 nocardia strains than the common antibiotics, and particularly, the MIC values of the alpha-mangostin are smaller than those of the antibiotics for aquatic products approved by agricultural departments such as enrofloxacin hydrochloride, levofloxacin hydrochloride, florfenicol or norfloxacin.
Examples
The embodiment provides an in-vivo alpha-mangostin injection for reducing bacterial copy number variation test of largehead jewfish infected with nocardia.
The Nocardia strain used in this example was NC61-AKLIL-0502. Healthy largemouth weever was selected, with an average body weight of 20±3.4g, and randomly divided into a control group, a negative control group, and a drug-treated group (different concentrations of α -mangostin). The drug treatment groups are respectively provided with three concentration treatment groups with low, medium and high concentration, each group is three parallel, and each group is provided with 20 parallel large-mouth black weever. The drug treatment group is injected with 50 mu L of alpha-mangostin injection (DMSO is solvent), and the drug concentration of the low, medium and high concentration treatment groups is 20mg/kg, 40mg/kg and 80mg/kg respectively. The control and negative control groups were injected with 50 μl of sterile PBS solution containing an equivalent amount of DMSO. Seven days after feeding, the negative control group was injected with 50 μl of sterile PBS solution, and each of the control group and the drug-treated group was intraperitoneally injected with 50 μl of half lethal concentration nocardia PBS suspension. 20mg of liver of Lateolabrax alopecuroides is taken after 4 days, DNA is extracted, and the content of B subunit gene of North Amyda Serratia DNA gyrase is determined by adopting a fluorescent quantitative PCR method (GeneBank accession No. SAP 017900.1). The primer sequences used were as follows: nsgyrb_f 5'-GATGTGGACACCGACGGATT-3', nsgyrB _r 5'-TGTTCAGGAACGCCATCTCC-3'.
The results of the nocardia copy number measurement of the liver of micropterus salmoides are shown in fig. 3. As can be seen from FIG. 3, the treatment of alpha-mangostin with different concentrations can significantly reduce the copy number of nocardia in the liver of Lateolabrax japonicusP<0.01 The copy number reduction factors of nocardia in the 20mg/kg treatment group, the 40mg/kg treatment group and the 80mg/kg treatment group are 5.24 times, 9.67 times and 8.13 times respectively.
Examples
The present example provides an experiment of alpha-mangostin for prevention and treatment of jewfish nocardia.
The Nocardia strain used in this example was NC61-AKLIL-0502, and the toxicity test was conducted to determine that the half-lethal concentration of Nocardia on Lateolabrax is 1×10 in 21 days 7 cfu/mL。
Healthy largemouth bass 540 tails are selected, the average weight is 20g plus or minus 2.0g, the treatment groups are randomly divided into 6 treatment groups, and 30 tails of each treatment are repeated, and the treatment groups are respectively a negative control group, a positive control group, a test group 1, a test group 2 and a test group 3. The control group and the negative control group are fed with compound feed (through weever compound feed), the test group 1 is fed with compound feed +200mg/kg alpha-mangostin (drug content/feed weight, i.e. the mass ratio of alpha-mangostin per milligram to the basic compound feed of per kilogram of largehead bass), the test group 2 is fed with compound feed +400mg/kg alpha-mangostin, the test group 3 is fed with compound feed +800mg/kg alpha-mangostin, and the positive control group is fed with compound feed +100mg/kg enrofloxacin hydrochloride 2 times per day.
On day 7, 100 μl of sterile PBS was intraperitoneally injected per fish in the negative control group, and 100 μl of half lethal concentration nocardia suspension prepared by sterile PBS was injected per fish in the control group, the positive control group, and the test group. On test day 14, 3 fish were randomly selected for liver collection, DNA was extracted to determine the copy number of nocardia, and survival rates of each group were counted for 21 days after challenge, and the results are shown in FIGS. 4 and 5.
As can be seen from FIG. 4, the addition levels of 400mg/kg of alpha-mangostin with respect to the nocardia content in 200. 200mg/kg, 400mg/kg and 800mg/kg of the addition groups were 5.74, 11.84 and 9.23 times, and the effect of inhibiting proliferation of nocardia in fish was best.
As can be seen from FIG. 5, when the alpha-mangostin provided by the invention is used for preventing and treating the largehead jewfish nocardia, the effect is obviously higher than that of a positive control group (enrofloxacin hydrochloride), wherein the addition level of 400mg/kg of the alpha-mangostin has the highest protection rate on the largehead jewfish.
Examples
The present example provides a nocardia soaking infection test for injecting alpha-mangostin for micropterus salmoides.
The Nocardia strain used in this example was NC61-AKLIL-0502. Healthy largemouth bass was selected, with an average body weight of 20.+ -. 2.0g, and randomized into control, negative and positive control (enrofloxacin hydrochloride) and drug-treated (alpha-mangostin). Each group had 3 parallels, 30 tails per parallel. Immersing each group of test fish into the bacterial liquid with the density of 2.1 multiplied by 10 7 In cfu/mL of water, the bacteria liquid is supplemented by daily water change to maintain the density. Positive control group was continued for 7 days at abdomenThe enrofloxacin hydrochloride solution prepared by injecting 50 mu L of DMSO solution into the cavity is injected into the cavity, the injection concentration is 40mg/k, the alpha-mangostin solution of 40mg/kg is injected into the abdominal cavity of a drug treatment group for 7 continuous days, the DMSO solvent of the same amount is injected into a negative control group for seven continuous days, and 0.1mg/L eugenol is used for carrying out anesthesia on the largehead jewfish before each operation in order to reduce stress. On day 14, 5 fish were dissected under aseptic conditions from each of the parallel animals, homogenates were prepared, gradient dilutions were applied to soy casein solid medium (tsb+15% agar), nocardia viable count was performed after incubation at 28 ℃ for 7 days, and infection rate was counted, infection rate = nocardia fish detected/total x 100%, survival rate was counted after 21 days, survival rate= (total number-death number)/total x 100%, and experimental results are shown in table 4.
TABLE 4 survival rate of largemouth bass in nocardia soaking infection test
Nocardia content (cfu/g) Infection rate (%) Number of deaths (tail) Survival (%)
Control group 1.7×10 2 ~1.9×10 3 100 68 24.4
Negative control group 1.7×10 2 ~1.9×10 3 100 67 25.5
Drug treatment group 0 0 0 100
Positive control group 0~1.8×10 2 40 26 71.1
As can be seen from Table 4, through the soaking infection experiment, the infection rate of the largehead jewfish injected with the alpha-mangostin is 0, the survival rate reaches 100%, which shows that the alpha-mangostin can effectively prevent the infection of nocardia in a natural state, thereby effectively preventing and treating the occurrence of nocardia in fish.
The present invention may be better implemented as described above, and the above examples are merely illustrative of preferred embodiments of the present invention and not intended to limit the scope of the present invention, and various changes and modifications made by those skilled in the art to the technical solution of the present invention should fall within the scope of protection defined by the present invention without departing from the spirit of the design of the present invention.

Claims (2)

1. Application of mangosteen extract, or alpha-mangostin, or beta-mangostin, or gamma-mangostin, or iso-mangostin in preparation of nocardia control drugs, and is characterized in that the nocardia control drugsThe fungus is Nocardia SeriolaN. seriolae；
The active ingredients of the mangosteen extract comprise at least one of alpha-mangostin, beta-mangostin, gamma-mangostin and iso-mangostin;
the effective components of the medicine are one or more of mangosteen extract, alpha-mangostin, beta-mangostin, gamma-mangostin and iso-mangostin.
2. The use according to claim 1, characterized in that the medicament is for the control of nocardia in fish.
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