Prevention and control method for orbiculasia serra based on artemisia apiacea extract
By using artemisia annua extract to control Helicobacter pylori infection, the cell structure and physiological function of Helicobacter pylori are disrupted, thus solving the problem of Helicobacter pylori infection and achieving a highly efficient and environmentally friendly control effect.
Patent Information
- Application Number
- CN202511033345.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-25
- Publication Date
- 2025-11-21
AI Technical Summary
Current technologies are ineffective in controlling infection with Helicobacter erythrozoa, leading to pathological changes and economic losses in crustaceans. The use of chemical drugs causes food safety and environmental pollution, and crustaceans lack acquired immunity, making it impossible to control the disease through vaccines or immunizing agents.
Artemisia annua extract was used to control Helicobacter pylori. By isolating the parasites and adding different concentrations of Artemisia annua extract to the culture medium, changes in mitochondrial membrane potential and ATPase activity were detected, and the effective concentration was screened to induce apoptosis in the parasites.
Artemisia annua extract significantly kills Helicobacter pylori, destroys its cell structure and physiological function, reduces the risk of drug resistance, meets the requirements of green farming, and leaves no harmful residues.
Smart Images
Figure CN120983501A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of pharmaceutical technology, and in particular to a method for controlling Helicobacter pylori based on Artemisia annua extract. Background Technology
[0002] *Hemiberleinii* is an obligate parasitic dinoflagellate that infects more than 70 important economically important marine crustaceans, including the swimming crab (Portunus trituberculatus), blue crab (Swiss blue crab), and Norwegian lobster. Infection with *Hemiberleinii* can cause severe pathological changes in crustacean hosts, such as "milk disease" (milky white body fluid) in swimming crabs and whiteleg shrimp, and "yellow water disease" (yellowish, cloudy hemolymph) in mud crabs, ultimately leading to host death and causing significant economic losses to marine crustacean aquaculture.
[0003] Plant extracts, due to their natural origin, low toxicity, lack of harmful residues, no withdrawal period, and abundant resources, have gradually become a research hotspot for disease control in aquaculture. Medicinal plants or their extracts can improve the health of farmed animals through multiple pathways, such as promoting growth, enhancing immunity, antibacterial activity, and anti-stress. Among them, artemisinin and its derivatives, as the main active ingredients of the traditional Chinese medicine Artemisia annua, have been proven to have significant inhibitory effects on various parasitic protozoa (such as Plasmodium, Toxoplasma gondii, and coccidia). Their mechanisms of action include: blocking the early absorption and metabolism of nutrients in the parasite, causing amino acid deficiency and cell membrane damage; binding to a wide range of parasitic proteins, interfering with key physiological processes such as hemoglobin endocytosis, glycolysis, protein synthesis, and the cell cycle; activating free Fe(II)PPIX to cleave internal peroxide bridges, generating carbon-centered free radicals and triggering a burst of cytotoxic reactive oxygen species (ROS); inducing mitochondrial membrane depolarization, promoting mitophagy, and ultimately leading to parasite death.
[0004] While chemical pesticides and antibiotics can control diseases in the short term, they easily lead to drug residues, food safety risks, and damage to the aquatic environment, which does not meet the development needs of green aquaculture. Economic crustaceans lack acquired immunity to Helicobacter pylori, and the development of existing vaccines or immunizing agents has not yet made breakthroughs, making it impossible to prevent infection through active immunization. When disease outbreaks occur, farmers mainly rely on large-scale application of antibiotics and pesticides for emergency control, but such methods not only fail to eradicate the pathogen but also exacerbate the pathogen's drug resistance and environmental pollution. Based on this, this invention experimentally verified the in vitro killing activity of Artemisia annua extract against Helicobacter pylori (including amoeba-like trophozoites and microspores), elucidated its destructive effects on the worm's cell structure (such as cell membrane damage and mitochondrial damage), physiological vitality (such as inhibition of ATP synthase activity), and the molecular mechanism of inducing apoptosis, providing a new approach for developing environmentally friendly Helicobacter pylori control drugs. Summary of the Invention
[0005] The purpose of this invention is to provide a method for controlling Helicobacter oryzae based on Artemisia annua extract, addressing the lack of effective control methods for Helicobacter oryzae epidemics mentioned above. Drug abuse damages food safety and aquatic ecosystems, failing to meet the development needs of green aquaculture. Furthermore, economically important crustaceans lack acquired immunity, making it impossible to obtain vaccines or immunizing agents for effective control of this parasitic dinoflagellate epidemic. During outbreaks, farmers mainly rely on large-scale application of antibiotics and pesticides for emergency control, but such methods not only fail to eradicate the pathogen but also exacerbate drug resistance and environmental pollution.
[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for controlling *Heliotropium indicum* based on *Artemisia annua* extract, the specific steps of which are as follows:
[0007] Step 1: Artemisia annua extract was purchased from Shanghai Yuanye Biotechnology Co., Ltd.
[0008] Step 2: Isolate Hemootrichum canaliculus from economically important crustaceans to obtain its amoeba-like trophozoites and microspores;
[0009] Step 3: The amoeba-like trophozoites are inoculated into a culture medium containing specific components, namely 0.1% penicillin and streptomycin, 5% serum from healthy swimming crabs, 10% fetal bovine serum and 85% Nephrops-Saline buffer, and the microspores are cultured using sterile filtered seawater.
[0010] Step 4: Add different concentrations of the Artemisia annua extract to the culture system for intervention, including concentrations of 1000 mg / L, 100 mg / L, and 10 mg / L;
[0011] Step 5: Collect parasites at 4h and 24h after intervention, and detect changes in mitochondrial membrane potential of *Helicobacter pylori* using a multifunctional ELISA reader, including the following steps:
[0012] (1) Take 500,000 cells and resuspend them in 0.5 ml of cell culture medium. The cell culture medium may contain serum and phenol red.
[0013] (2) Add 0.5 ml of JC-1 staining working solution and mix by inverting several times. Incubate at 37°C for 20 minutes in a cell culture incubator.
[0014] (3) During incubation, prepare an appropriate amount of JC-1 staining buffer (1×) by adding 4 ml of distilled water to every 1 ml of JC-1 staining buffer (5×) and place it in an ice bath.
[0015] (4) After incubation at 37℃, centrifuge at 600g at 4℃ for 3-4 minutes to precipitate the cells. Discard the supernatant, being careful not to aspirate the cells.
[0016] (5) Wash twice with JC-1 staining buffer (1×): Add 1 ml of JC-1 staining buffer (1×) to resuspend the cells, centrifuge at 600 g at 4℃ for 3-4 minutes, precipitate the cells, and discard the supernatant.
[0017] (6) After resuspending with an appropriate amount of JC-1 staining buffer (1×), analyze with a multi-functional microplate reader.
[0018] Step 6: Based on the test results, screen out the concentration of Artemisia annua extract that has a significant killing effect on the amoeba-like trophozoites and microspores of Helicobacter pylori.
[0019] Preferably, the economically important crustaceans include at least one of the swimming crab (Portunus trituberculatus) and the mud crab (Scylla serrata).
[0020] Preferably, the method for isolating Helicobacter ovalis is as follows: after inoculating the fifth appendage of a Tianjin thick crab naturally infected with Helicobacter ovalis for 21 to 40 days, microspores of Helicobacter ovalis are isolated, and amoeba-like trophozoites are isolated and purified from the hemolymph of the Tianjin thick crab infected with Helicobacter ovalis.
[0021] The results showed that after treatment with 1000 mg / L Artemisia annua extract, the proportion of JC-1 monomers in the amoeba-like trophozoites of Helicobacter erythrocystis significantly increased, and the mitochondrial membrane potential significantly decreased. After treatment with 100 mg / L and 1000 mg / L Artemisia annua extracts, the proportion of JC-1 monomers in the microspores of Helicobacter erythrocystis significantly increased, the mitochondrial membrane potential significantly decreased, and apoptosis occurred.
[0022] Preferably, the damaging effect of the artemisia annua extract on *Heliotropium indicum* is confirmed by transmission electron microscopy, specifically as follows:
[0023] (1) The cell membrane is damaged, and the cytoplasm is severely lost;
[0024] (2) The cells swell and vacuolated structures appear;
[0025] (3) Mitochondria swell and become round, and the internal crest structure breaks or even disappears.
[0026] Preferably, the effect of the artemisia annua extract on the physiological viability of *Heliotropium indicum* cells is detected by measuring mitochondrial H... + The determination of ATPase activity includes the following specific steps:
[0027] (1) After collecting the cells, wash them twice with PBS (1000 rpm, 15 min) and transfer them to 1.5 mL centrifuge tubes;
[0028] (2) Preparation of mitochondria at 4℃: Add 1 mL of extraction solution 1 to the cells, homogenize in an ice bath, centrifuge at 3000g for 20 min at 4℃, and take the supernatant; centrifuge at 16000g for 20 min at 4℃, and the precipitate is the mitochondria;
[0029] (3) Add 200 μL of extraction solution 2 to the mitochondria and sonicate them in an ice bath (power 200W, sonication for 5s, interval 3s, repeated 30 times).
[0030] (4) According to the mitochondrial H of youyi-1359 + - ATPase kit instructions for detecting ATP synthase activity;
[0031] (5) Results: After treatment with Artemisia annua extract, the ATP synthase activity of the amoeba-like trophozoites was significantly reduced, leading to cell necrosis. The ATP synthase activity of the microspores was significantly reduced at 4 h and significantly increased at 24 h, triggering apoptosis.
[0032] Compared with the prior art, the beneficial effects of the present invention are:
[0033] 1. Artemisia annua extract has a significant killing effect on Helicobacter pylori (including amoeba-like trophozoites and microspores), and can induce apoptosis and necrosis of the parasite, destroying its cell structure and physiological function.
[0034] 2. It achieves high-efficiency prevention and control through multi-target action (such as disrupting mitochondrial function and inducing oxidative damage), reduces the risk of drug resistance, and has natural ingredients with no toxic side effects;
[0035] 3. Derived from the plant Artemisia annua, it has no harmful residues and no withdrawal period, avoiding chemical drug pollution and meeting the needs of green farming.
[0036] Figure 1 This is a transmission electron microscope schematic diagram of the amoeba-like trophozoites and microspores of *Helicobacter pylori* after administration of the artemisia extract of the present invention.
[0037] Figure 2 This is a schematic diagram showing the electrical changes of the amoeba-like trophozoites and microspore membranes of Helicobacter pylori after administration of Artemisia annua extract according to the present invention.
[0038] Figure 3 This is a schematic diagram showing the effect of Artemisia annua extract of the present invention on the ATP synthase activity of amoeba-like trophozoites and microspores of Helicobacter pylori. Detailed Implementation
[0039] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0040] Please see Figure 1-3 This invention provides a technical solution: a method for controlling Helicobacter annuus based on Artemisia annua extract, the specific steps of which are as follows:
[0041] Step 1: Artemisia annua extract was purchased from Shanghai Yuanye Biotechnology Co., Ltd.
[0042] Step 2: Isolate Hemootrichum canaliculus from economically important crustaceans to obtain its amoeba-like trophozoites and microspores;
[0043] Step 3: Inoculate the amoeba-like trophozoites and microspores separately into a culture medium containing specific components, namely 0.1% penicillin and streptomycin, 5% serum from healthy swimming crabs, 10% fetal bovine serum and 85% Nephrops-Saline buffer, and culture the microspores in sterile filtered seawater.
[0044] Step 4: Add different concentrations of Artemisia annua extract to the culture system for intervention, including 1000 mg / L, 100 mg / L, and 10 mg / L;
[0045] Step 5: Collect parasites at 4h and 24h after intervention, and detect changes in mitochondrial membrane potential of *Helicobacter pylori* using a multifunctional ELISA reader, including the following steps:
[0046] (1) Take 500,000 cells and resuspend them in 0.5 ml of cell culture medium. The cell culture medium may contain serum and phenol red.
[0047] (2) Add 0.5 ml of JC-1 staining working solution and mix by inverting several times. Incubate at 37°C for 20 minutes in a cell culture incubator.
[0048] (3) During incubation, prepare an appropriate amount of JC-1 staining buffer (1×) by adding 4 ml of distilled water to every 1 ml of JC-1 staining buffer (5×) and place it in an ice bath.
[0049] (4) After incubation at 37℃, centrifuge at 600g at 4℃ for 3-4 minutes to precipitate the cells. Discard the supernatant, being careful not to aspirate the cells.
[0050] (5) Wash twice with JC-1 staining buffer (1×): Add 1 ml of JC-1 staining buffer (1×) to resuspend the cells, centrifuge at 600 g at 4℃ for 3-4 minutes, precipitate the cells, and discard the supernatant.
[0051] (6) After resuspending with an appropriate amount of JC-1 staining buffer (1×), analyze with a multi-functional microplate reader.
[0052] Furthermore, economically important crustaceans include at least one of the swimming crab (Portunus trituberculatus) and the mud crab (Scylla serrata).
[0053] Furthermore, the method for isolating Helicobacter erythrocystis is as follows: after inoculating the fifth appendage of a Tianjin thick crab naturally infected with Helicobacter erythrocystis for 21 to 40 days, microspores of Helicobacter erythrocystis are isolated, and amoeba-like trophozoites are isolated and purified from the hemolymph of Helicobacter erythrocystis infected with Helicobacter erythrocystis.
[0054] Furthermore, the intervention of artemisia annua extract includes its in vitro killing effect on microspores and trophozoites of Helicobacter pylori, specifically manifested as follows:
[0055] Treatment with 1000 mg / L Artemisia annua extract significantly increased the proportion of JC-1 monomers in the amoeba-like trophozoites of Helicobacter erythropoietin, and significantly decreased the mitochondrial membrane potential. Treatment with 100 mg / L and 1000 mg / L Artemisia annua extracts significantly increased the proportion of JC-1 monomers in the microspores of Helicobacter erythropoietin, significantly decreased the mitochondrial membrane potential, and induced apoptosis.
[0056] Furthermore, the damaging effect of Artemisia annua extract on Helicobacter pylori was confirmed by transmission electron microscopy, specifically as follows:
[0057] (1) Cell membrane damage and severe loss of cytoplasm;
[0058] (2) Cells swell and vacuolated structures appear;
[0059] (3) Mitochondria swell and become round, and the inner crest structure breaks or even disappears.
[0060] Furthermore, the effect of artemisia annua extract on the physiological viability of *Heliotropium indicum* cells was investigated by detecting mitochondrial H... + The determination of ATPase activity includes the following specific steps:
[0061] (1) After collecting the cells, wash them twice with PBS (1000 rpm, 15 min) and transfer them to 1.5 mL centrifuge tubes;
[0062] (2) Preparation of mitochondria at 4℃: Add 1mL of extraction solution 1 to the cells, homogenize in an ice bath, centrifuge at 3000g for 20min at 4℃, and take the supernatant; centrifuge at 16000g for 20min at 4℃, and the precipitate is the mitochondria;
[0063] (3) Add 200 μL of extraction solution 2 to the mitochondria and sonicate them in an ice bath (power 200W, sonication for 5s, interval 3s, repeated 30 times).
[0064] (4) According to the mitochondrial H of youyi-1359 + - ATPase kit instructions for detecting ATP synthase activity;
[0065] (5) Result determination: After treatment with Artemisia annua extract, the ATP synthase activity of the amoeba-like trophozoites was significantly reduced, leading to cell necrosis. The ATP synthase activity of the microspores was significantly reduced at 4h and significantly increased at 24h, triggering apoptosis.
[0066] Example 1: Observation of the microscopic changes of *Heliotropium indicum* under the action of different concentrations of *Artemisia annua* extract for 24 hours, to explore the degree of damage and effect of *Artemisia annua* extract on *Heliotropium indicum*, specifically:
[0067] (1) Cell collection: Aseptically isolated amoeba-like trophozoites of *Hymenococcus ovalis* were placed in 6-well plates containing 300 μL of culture medium (0.1% penicillin-streptomycin + 5% healthy *Portunus trituberculatus* serum + 10% fetal bovine serum + 85% Nephrops-Saline buffer) and diluted to 10⁻⁶. 6 cells / mL; Microspores naturally released by Tianjin thick crab were placed in a 6-well plate containing 300 μL of sterile filtered seawater and diluted to 10⁻⁶ cells / mL. 6 Different concentrations of Artemisia annua extract were added to the culture system for intervention. The experimental groups were set as Artemisia annua extract treatment group (1000 mg / L, 100 mg / L) and blank control group. The insect bodies were collected at 4 h and 24 h after administration, and the changes in cell ultrastructure were observed by transmission electron microscopy. The specific steps are as follows: The collected cells (collection method is the same as in Experiment 1) were washed twice with PBS (1000 rpm, 15 min). After washing, they were transferred to 1.5 mL centrifuge tubes.
[0068] (2) At room temperature, add 1 mL of 2.5% glutaraldehyde solution to the cell pellet for fixation and store at 4°C;
[0069] (3) 1% osmium tetroxide was prepared in 0.1M phosphate buffer PB (pH 7.4) and fixed at room temperature in the dark for 2 hours. It was then rinsed 3 times with 0.1M phosphate buffer PB (pH 7.4) for 15 minutes each time.
[0070] (4) Room temperature dehydration: The tissue was sequentially immersed in 30%-50%-70%-80%-95%-100%-100% alcohol for upward dehydration, each time for 20 minutes, and then in 100% acetone twice, each time for 15 minutes;
[0071] (5) Infiltration embedding: Acetone: 812 embedding agent = 1:1 and 37℃ for 2h, acetone: 812 embedding agent = 1:2 and infiltration overnight at 37℃, pure 812 embedding agent at 37℃ for 5h, pour pure 812 embedding agent into embedding plate, insert the sample into embedding plate and bake at 37℃ overnight.
[0072] (6) Polymerization: The embedding plate was placed in a 60℃ oven for polymerization for 48 hours, and the resin block was removed for later use;
[0073] (7) Positioning: The resin block was sectioned into 1.5μm sections using a microtome, stained with toluidine blue, and positioned under a light microscope;
[0074] (8) Ultrathin sectioning: The resin block is used to make ultrathin sections of 60-80nm using an ultrathin slicer, and the sections are retrieved using a 150-mesh copper mesh film.
[0075] (9) Staining: The copper mesh was stained in 2% uranium acetate saturated alcohol solution for 8 min in the dark, washed 3 times with 70% alcohol, washed 3 times with ultrapure water, stained in 2.6% lead citrate solution for 8 min in the dark, washed 3 times with ultrapure water, slightly dried with filter paper, and the copper mesh sections were placed in a copper mesh box to dry overnight at room temperature.
[0076] (10) Observe under a transmission electron microscope and collect and analyze images.
[0077] The results are as follows Figure 1 As shown, after drug treatment, the cell membrane of *Helicobacter erythrozoa* was damaged, cytoplasm was severely lost, cells swelled, and vacuolated structures appeared; mitochondria swelled and became rounded, and the cristae structure broke or even disappeared.
[0078] Example 2: The decrease in mitochondrial membrane potential is a hallmark event in the early stages of apoptosis. This decrease can be easily detected by monitoring the transition of JC-1 from red to green fluorescence, making it an early indicator of apoptosis. The specific steps are as follows:
[0079] (1) Take 500,000 cells and resuspend them in 0.5 ml of cell culture medium. The cell culture medium may contain serum and phenol red.
[0080] (2) Add 0.5 ml of JC-1 staining working solution and mix by inverting several times. Incubate at 37°C for 20 minutes in a cell culture incubator.
[0081] (3) During incubation, prepare an appropriate amount of JC-1 staining buffer (1×) by adding 4 ml of distilled water to every 1 ml of JC-1 staining buffer (5×) and place it in an ice bath.
[0082] (4) After incubation at 37℃, centrifuge at 600g at 4℃ for 3-4 minutes to precipitate the cells. Discard the supernatant, being careful not to aspirate the cells.
[0083] (5) Wash twice with JC-1 staining buffer (1×): Add 1 ml of JC-1 staining buffer (1×) to resuspend the cells, centrifuge at 600 g at 4℃ for 3-4 minutes, precipitate the cells, and discard the supernatant.
[0084] (6) After resuspending with an appropriate amount of JC-1 staining buffer (1×), analyze with a multi-functional microplate reader.
[0085] The results are as follows Figure 2 As shown, after treatment with 1000 mg / L Artemisia annua extract, the proportion of JC-1 monomers in the amoeba-like trophozoites of Helicobacter erythrocyst significantly increased, and the mitochondrial membrane potential significantly decreased; after treatment with 100 mg / L and 1000 mg / L Artemisia annua extract, the proportion of JC-1 monomers in the microspores of Helicobacter erythrocyst significantly increased, the mitochondrial membrane potential significantly decreased, and apoptosis occurred.
[0086] Example 3: Effects of Artemisia annua extract on the physiological viability of Helicobacter pylori cells. Mitochondria are important sites of cellular respiration and metabolism, and H2O located in the inner mitochondrial membrane... + - ATPase is a key component of oxidative phosphorylation coupling and a crucial component in ATP synthesis. ATP synthase activity can reflect cell viability. The specific experimental steps are as follows:
[0087] (1) The collected cells (collected in the same way as in Experiment 1) were washed twice with PBS (1000 rpm, 15 min) and then transferred to a 1.5 mL centrifuge tube.
[0088] (2) The experiment used Youyi-1359 mitochondrial H + - ATPase kit, mitochondrial preparation (Note: The entire mitochondrial extraction process must be kept at a low temperature of 4℃): Add 1 mL of extraction solution 1 to the collected cells, homogenize in a mortar on ice, transfer to a centrifuge tube, and centrifuge at 4℃×3000g for 20 min. Carefully aspirate the supernatant (discard the precipitate) and transfer it to another centrifuge tube. Centrifuge at 4℃×16000g for 20 min, leaving the precipitate (the precipitate is the mitochondria). Add 200 μL of extraction solution 2 to the precipitate (mitochondria), and sonicate (ice bath, power 200W, sonication for 5 s, interval 3 s, repeated 30 times). Place the liquid on ice for mitochondrial H2O. + -ATPase activity assay;
[0089] (3) Add the experimental reagents in sequence according to the kit instructions and prepare for detection with an ELISA reader.
[0090] The results are as follows Figure 3As shown, after treatment with Artemisia annua extract, the ATP synthase activity of amoeba-like trophozoites was significantly reduced, leading to cell necrosis. The ATP synthase activity of microspores was significantly reduced at 4 h and significantly increased at 24 h, inducing the cell's energy emergency response compensation mechanism and triggering apoptosis.
[0091] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.
Claims
1. A method for controlling *Heliotropium indicum* based on *Artemisia annua* extract, characterized in that, The specific steps are as follows: Step 1: Artemisia annua extract was purchased from Shanghai Yuanye Biotechnology Co., Ltd. Step 2: Isolate Hemootrichum canaliculus from economically important crustaceans to obtain its amoeba-like trophozoites and microspores; Step 3: The amoeba-like trophozoites are inoculated into a culture medium containing specific components, namely 0.1% penicillin and streptomycin, 5% serum from healthy swimming crabs, 10% fetal bovine serum and 85% Nephrops-Saline buffer, and the microspores are cultured using sterile filtered seawater. Step 4: Add different concentrations of the Artemisia annua extract to the culture system for intervention, including concentrations of 1000 mg / L, 100 mg / L, and 10 mg / L; Step 5: After intervention, parasites were collected at 4h and 24h. Changes in mitochondrial membrane potential of *Helicobacter pylori* were detected using a multifunctional ELISA reader, and the microstructure of the parasites was observed using transmission electron microscopy. Mitochondrial H... + -ATPase activity; Step Six: Based on the mitochondrial membrane potential experiment and mitochondrial H... + -ATPase activity assay and transmission electron microscopy results were used to screen the concentration of Artemisia annua extract that has a significant killing effect on the amoeba-like trophozoites and microspores of Helicobacter pylori.
2. The method for controlling Helicobacter pylori based on Artemisia annua extract according to claim 1, characterized in that: The economically important crustaceans include at least one of the swimming crab (Portunus trituberculatus) and the mud crab (Scylla serrata).
3. The method for controlling Helicobacter pylori based on Artemisia annua extract according to claim 1, characterized in that: The method for isolating Helicobacter ovalis is as follows: after inoculating the fifth appendage of a Tianjin thick crab naturally infected with Helicobacter ovalis for 21 to 40 days, microspores of Helicobacter ovalis are isolated, and amoeba-like trophozoites are isolated and purified from the hemolymph of the Tianjin thick crab infected with Helicobacter ovalis.
4. The method for controlling Helicobacter pylori based on Artemisia annua extract according to claim 1, characterized in that: The intervention of the Artemisia annua extract includes an in vitro killing effect on the microspores and trophozoite stages of Helicobacter pylori, specifically manifested as follows: Treatment with 1000 mg / L Artemisia annua extract significantly increased the proportion of JC-1 monomers in the amoeba-like trophozoites of Helicobacter erythropoietin, and significantly decreased the mitochondrial membrane potential. Treatment with 100 mg / L and 1000 mg / L Artemisia annua extracts significantly increased the proportion of JC-1 monomers in the microspores of Helicobacter erythropoietin, significantly decreased the mitochondrial membrane potential, and induced apoptosis.
5. The method for controlling Helicobacter pylori based on Artemisia annua extract according to claim 1, characterized in that: The damaging effect of the artemisia annua extract on *Heliotropium indicum* was confirmed by transmission electron microscopy, specifically as follows: (1) The cell membrane is damaged, and the cytoplasm is severely lost; (2) The cells swell and vacuolated structures appear; (3) Mitochondria swell and become round, and the internal crest structure breaks or even disappears.
6. The method for controlling Helicobacter pylori based on Artemisia annua extract according to claim 1, characterized in that: The effect of Artemisia annua extract on the physiological viability of Helicobacter pylori cells was determined by detecting mitochondrial H. + The determination of ATPase activity includes the following specific steps: (1) After collecting the cells, wash them twice with PBS (1000 rpm, 15 min) and transfer them to 1.5 mL centrifuge tubes; (2) Preparation of mitochondria at 4℃: Add 1 mL of extraction solution 1 to the cells, homogenize in an ice bath, centrifuge at 3000g for 20 min at 4℃, and take the supernatant; centrifuge at 16000g for 20 min at 4℃, and the precipitate is the mitochondria; (3) Add 200 μL of extraction solution 2 to the mitochondria and sonicate them in an ice bath (power 200W, sonication for 5s, interval 3s, repeated 30 times). (4) According to the mitochondrial H of youyi-1359 + - ATPase kit instructions for detecting ATP synthase activity; (5) Results: After treatment with Artemisia annua extract, the ATP synthase activity of the amoeba-like trophozoites was significantly reduced, leading to cell necrosis. The ATP synthase activity of the microspores was significantly reduced at 4 h and significantly increased at 24 h, triggering apoptosis.