Application of sea-buckthorn extract in killing trichomonad pigeon and preparation method of sea-buckthorn extract

Abstract

The invention discloses application of a sea-buckthorn extract to killing in-vitro cultured trichomonad pigeon and a preparation method of the sea-buckthorn extract, and belongs to the technical field of traditional Chinese medicines and the field of animal disease control, the sea-buckthorn extract is prepared by the following preparation method: crushing dried and ripe sea-buckthorn fruits, performing condensation reflux extraction by using 70% ethanol, and collecting an extracting solution; concentrating by using a rotary evaporator; drying in an electrothermal blowing drying box at 55 DEG C to obtain an extract; according to the method, the effective insecticidal components of the sea-buckthorn are extracted, and the method is easy to implement and convenient to operate; the alcohol extract is diluted according to a certain proportion and then used for killing trichomonad pigeon cultured in vitro, and the insecticidal effect is remarkable; compared with traditional chemical drugs, the alcohol extract prepared from the sea-buckthorn as the raw material is used for preventing and controlling trichomonad of pigeons, has the advantages of no residue, no drug resistance, greenness and safety, and has great popularization value.

Description

Technical Field

[0001] The present invention belongs to the technical fields of traditional Chinese medicine and animal disease prevention and control, and specifically relates to a seabuckthorn extract mainly made of seabuckthorn and its effect on killing Trichomonas gallinae cultured in vitro. Background Art

[0002] Trichomoniasis in pigeons is a very common and highly harmful parasitic disease caused by Trichomonas gallinae, which is widely spread globally and seriously endangers the development of the pigeon breeding industry. Trichomonas gallinae usually parasitizes in parts such as the oral cavity, pharynx, esophagus and crop of pigeons, and is transmitted through direct contact or indirect contact (including contaminated feed and water sources). The mechanism of its infection and pathogenesis is that Trichomonas gallinae will use its unique pseudopodia and undulating membrane to shuttle between the epithelial cells of pigeons, destroy the cell structure, and trigger an inflammatory response. In the initial stage, pigeons may only show symptoms such as slightly increased oral mucus and loss of appetite, but as the disease progresses, there will be serious consequences such as listlessness, ruffled feathers, retarded growth and development, and even death, which not only greatly damages the health of pigeons, but also has a huge impact on the stability and breeding efficiency of the entire breeding industry.

[0003] At present, the treatment of trichomoniasis in pigeons mainly relies on chemical drugs, including nitroimidazoles such as metronidazole and antibiotic drugs. However, the disadvantages of frequent long-term use of chemical drugs are obvious. First, it will make Trichomonas gallinae develop drug resistance, reduce the treatment effect, and increase the treatment difficulty and cost; second, the drug residues in the pigeons' bodies will damage the human nervous system and immune system when consumed by humans, endangering human health; third, the drug enters the environment with excrement, is difficult to degrade, and destroys the ecological balance. Therefore, it is of great significance to develop safe, highly effective and residue-free anti-Trichomonas gallinae drugs. Traditional Chinese medicine has become a research hotspot and new direction for the treatment of trichomoniasis in pigeons due to its advantages of natural ingredients, small side effects, multi-target effects and low drug resistance. Summary of the Invention

[0004] The purpose of the present invention is to overcome the above-mentioned defects in the prior art, provide a seabuckthorn alcohol extract mainly made of seabuckthorn and its preparation method, and explore the inhibitory and killing effects of the seabuckthorn alcohol extract on Trichomonas gallinae under in vitro culture conditions. The results show that the seabuckthorn extract can effectively inhibit and kill Trichomonas gallinae cultured in vitro, providing a reference for the later research on anti-Trichomonas gallinae traditional Chinese medicine and its application in animal breeding.

[0005] To achieve the above object, the present invention provides a seabuckthorn alcohol extract with seabuckthorn as the main raw material. The seabuckthorn extract mainly includes the following components: Vitamins: Seabuckthorn fruits are rich in vitamin C, vitamin E, folic acid and carotenoids. Among them, the content of vitamin C is between 750-1430 mg / 100 g, and the content of carotenoids is 34.1 mg / 100 g.

[0006] Flavonoids: The seabuckthorn extract contains various flavonoids, including isorhamnetin, quercetin and kaempferol; these flavonoids have antioxidant and anti-inflammatory effects.

[0007] Polysaccharides: Seabuckthorn polysaccharides are mainly composed of mannose, rhamnose, glucose, galactose and arabinose, and their contents are 12.73%, 5.86%, 26.71%, 6.3% and 48.41% respectively.

[0008] Polyphenolic compounds: The seabuckthorn extract is rich in various polyphenolic chemicals, including gallic acid, protocatechuic acid, caffeic acid and ferulic acid, and these substances have antioxidant and anti-inflammatory effects.

[0009] Protein: Seabuckthorn is rich in protein, especially the protein content in seabuckthorn seeds is between 25% and 28%.

[0010] In addition, the seabuckthorn extract also contains other bioactive components, including carotenoids, fatty acids, organic acids and sugars; these components together endow the seabuckthorn extract with various health effects, including antioxidant, anti-inflammatory, enhancing immune function and protecting cardiovascular health, etc.

[0011] The present invention provides a seabuckthorn extract, which is mainly prepared from the following materials by weight: 10 g of seabuckthorn fruits.

[0012] The preparation method of the above seabuckthorn extract is as follows. First, crush the seabuckthorn fruits with a crusher, sieve them through a 60-mesh sieve after crushing. Take a clean round-bottom flask, weigh 5-10 g of seabuckthorn powder, add 20-30 times the amount of 50%-70% ethanol solution, let it stand for 30 min, carry out condensation reflux at 50 °C for 50 min, then centrifuge at 7500 r for 10 min to collect the supernatant. Add 20-30 times the amount of 50%-70% ethanol to the centrifuged precipitate, carry out condensation reflux again for 30 min and then collect the supernatant, and combine the supernatants collected twice. Filter the collected supernatant by suction and concentrate the extract into a paste with a rotary evaporator. Add 5-10 ml of distilled water to ultrasonically dissolve the paste, collect it with a pre-weighed centrifuge tube, dry it in an electric thermostatic blast drying oven at 55 °C and then weigh it again, calculate the amount of extract and take it out and store it at -80 °C for standby.

[0013] As a further improvement of the present invention, for the preparation method of the seabuckthorn extract, first, the seabuckthorn fruits are crushed by a crusher, and after crushing, they are sieved through a 60-mesh sieve. Take a clean round-bottom flask and weigh 10 g of seabuckthorn powder, add 30 times the amount of 70% ethanol solution, let it stand for 30 min, carry out condensation reflux at 50 °C for 50 min, then centrifuge at 7500 r for 10 min to collect the supernatant. The precipitate from centrifugation is continuously added with 30 times the amount of 70% ethanol, and after condensation reflux for another 30 min, the supernatant is collected. The supernatants collected twice are combined. The collected supernatant is subjected to suction filtration, and the extract is concentrated into a paste by a rotary evaporator. Add 10 ml of distilled water and ultrasonically dissolve the paste, collect it with a pre-weighed centrifuge tube, dry it in an electrothermal blast drying oven at 55 °C and then weigh it again, calculate the amount of extract paste, take it out and store it at -80 °C for standby.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] 1. The insecticidal effect of the present invention is obvious. Through experimental verification, the traditional Chinese medicine extract of the present invention has an obvious killing effect on Trichomonas gallinae cultured in vitro.

[0016] 2. As a pure Chinese herbal medicine, the present invention has the characteristics of natural raw materials, safety, no residue, and no drug resistance. After medication, there will be no drug residue, which will not affect the quality of pigeon products and ensure the health of consumers. It is also not easy for Trichomonas gallinae to develop drug resistance and can be used stably for a long time.

[0017] 3. The preparation method of the seabuckthorn extract provided by the present invention is simple, and the obtained extract has a high yield, which can effectively reduce costs. Description of the Drawings

[0018] Figure 1 It is a graph showing the change of the mortality rate of Trichomonas gallinae cultured in vitro of the present invention with time under the action of seabuckthorn extracts at different concentrations. Detailed Embodiments

[0019] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0020] Example 1

[0021] The present invention provides a seabuckthorn extract, which is mainly prepared from the following materials by weight: 10 g of seabuckthorn fruits, 30 times the amount of 70% ethanol solution.

[0022] A preparation method of seabuckthorn extract in Example 1 of the present invention. First, crush seabuckthorn fruits with a crusher, sieve through a 60-mesh sieve after crushing. Take a clean round-bottom flask, weigh 10 g of seabuckthorn powder, add 30 times the amount of 70% ethanol solution, let it stand for 30 min, carry out condensation reflux at 50 °C for 50 min, then centrifuge at 7500 r for 10 min to collect the supernatant. Add 30 times the amount of 70% ethanol solution to the centrifuged precipitate again, carry out condensation reflux for 30 min again and then collect the supernatant. Combine the supernatants collected twice. Filter the collected supernatant by suction and concentrate the extract into a paste with a rotary evaporator. Add 10 ml of distilled water to ultrasonically dissolve the paste, collect it with a pre-weighed centrifuge tube, dry it in a vacuum drying oven and then weigh it again, calculate the extract yield and take it out. Store it at -80 °C for later use.

[0023] Among them, the extract yield is: (weight of the extract paste after drying / weight of the crude drug) × 100%.

[0024] If the extract yield is 50%, and the alcohol extract of 10 g of crude drug is 5 g of extract paste after drying, then 1 g of extract paste is equivalent to 2 g of crude drug.

[0025] The seabuckthorn extract yield is 42.68%.

[0026] Example 2

[0027] The present invention provides a seabuckthorn extract, which is mainly prepared from the following materials by weight: 10 g of seabuckthorn fruits, 30 times the amount of 60% ethanol solution.

[0028] Its preparation method is the same as that of Example 1 except for the different ethanol concentration.

[0029] Example 3

[0030] The present invention provides a seabuckthorn extract, which is mainly prepared from the following materials by weight: 5 g of seabuckthorn fruits, 30 times the amount of 60% ethanol solution.

[0031] Its preparation method is the same as that of Example 1 except for the different raw material quality and ethanol concentration.

[0032] Example 4

[0033] The present invention provides a seabuckthorn extract, which is mainly prepared from the following materials by weight: 5 g of seabuckthorn fruits, 30 times the amount of 70% ethanol solution.

[0034] Its preparation method is the same as that of Example 1 except for the different raw material quality.

[0035] Example 5

[0036] In order to clarify the effect of the seabuckthorn alcohol extract in Example 1 of the present invention on killing Trichomonas gallinae in vitro culture, the seabuckthorn alcohol extract in Example 1 was used as the experimental group for the anti-Trichomonas gallinae experiment on Trichomonas gallinae.

[0037] Experimental method of the ethanol extract of Hippohgae rhamnoides L. against Trichomonas gallinae in vitro culture, including the following steps:

[0038] S1. Culture of Trichomonas gallinae: Put the collected oral samples of diseased pigeons into HF complete medium and culture them in an incubator at 37 °C. Subculture once every 24 h with HF complete medium. After stable culture for 3 generations or more, use them for experiments;

[0039] S2. Concentration ratio of traditional Chinese medicine: Dilute the prepared concentration of Hippohgae rhamnoides L. crude drug at 400 mg / mL in a serial dilution with HF medium to prepare drug-containing media with crude drug concentrations of 400 mg / mL, 200 mg / mL, 100 mg / mL, 50 mg / mL, 25 mg / mL, 12.5 mg / mL, 6.25 mg / mL, and 3.125 mg / mL;

[0040] Concentration of crude drug: Mass / volume of crude drug after conversion of the extract ratio;

[0041] S3. Anti-insect treatment: Prepare a live worm suspension with a density of 2.5×10 5 per mL. Set two replicates for each concentration as the experimental group, 4 groups of metronidazole solution (final concentration of 50 μg / mL) as the positive control group, and 4 groups of double-distilled water as the negative control group. Add 120 μL of worm solution and 80 μL of traditional Chinese medicine solution (add 80 μL of metronidazole solution in the positive control group and 80 μL of double-distilled water in the negative control group) to each of the above tubes, that is, the drug-containing media with final crude drug concentrations of 160 mg / mL, 80 mg / mL, 40 mg / mL, 20 mg / mL, 10 mg / mL, 5 mg / mL, 2.5 mg / mL, and 1.25 mg / mL. Mix well and culture at 37 °C for 24 h;

[0042] S4. Effect observation: Select each time period of 6 h, 12 h, 18 h, 24 h, and 48 h during the culture process, observe the killing situation of the worms with a 400-fold optical microscope, and record the survival number of Trichomonas in each group. To ensure accurate results, for the group where all the worms are dead, transfer them normally (inoculate HF medium) and continue to culture for 24 h. If no live worms are determined, it can be judged that all the worms are killed at this concentration.

[0043] Results and analysis

[0044] It can be seen from Table 1 that the insecticidal effect of the Hippohgae rhamnoides L. extract reaches 98.7% at a final crude drug concentration of 10 mg / mL after 24 h and 100.0% after 48 h. From the perspective of the drug action time and effect observation, the insecticidal effect after 48 h is better than that after 24 h, indicating that prolonging the drug action time can improve the insecticidal effect.

[0045] Table 1 Observation of insecticidal effects at different drug concentrations

[0046]

[0047] Note: - indicates that the number of well-growing worms is close to or exceeds the inoculation number.

[0048] It should be noted that, in this document, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, such that a process, method, article or device comprising a series of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article or device.

[0049] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A seabuckthorn extract, characterized in that, It is mainly prepared from the following traditional Chinese medicines by weight: 5-10 g of seabuckthorn fruit.

2. A seabuckthorn extract according to claim 1, characterized in that, It is mainly prepared from the following traditional Chinese medicines by weight: 10 g of seabuckthorn fruit.

3. A method for preparing a seabuckthorn extract, based on the seabuckthorn extract described in claims 1-2, characterized in that, First, crush the seabuckthorn fruit with a crusher, sieve it through a 60-mesh sieve after crushing. Take a clean round-bottom flask, weigh 5-10 g of seabuckthorn powder, add 30 times the amount of 60%-70% ethanol solution, let it stand for 30 min, carry out reflux condensation at 50 °C for 50 min, then centrifuge at 7500 r for 10 min to collect the supernatant. Add 30 times the amount of 60%-70% ethanol solution to the centrifuged precipitate again, carry out reflux condensation for 30 min and then collect the supernatant. Combine the supernatants collected twice; filter the collected supernatant by suction and concentrate the extract into a paste with a rotary evaporator. Add 5-10 ml of distilled water to ultrasonically dissolve the paste, collect it with a pre-weighed centrifuge tube, dry it in an electric blast drying oven at 55 °C and then weigh it again. Calculate the amount of extract, take it out and store it at -80 °C for standby.

4. The preparation method of a seabuckthorn extract according to claim 3, wherein, First, crush the seabuckthorn fruit with a crusher, sieve it through a 60-mesh sieve after crushing. Take a clean round-bottom flask, weigh 10 g of seabuckthorn powder, add 30 times the amount of 70% ethanol solution, let it stand for 30 min, carry out reflux condensation at 50 °C for 50 min, then centrifuge at 7500 r for 10 min to collect the supernatant. Add 30 times the amount of 70% ethanol solution to the centrifuged precipitate again, carry out reflux condensation for 30 min and then collect the supernatant. Combine the supernatants collected twice; filter the collected supernatant by suction and concentrate the extract into a paste with a rotary evaporator. Add 5-10 ml of distilled water to ultrasonically dissolve the paste, collect it with a pre-weighed centrifuge tube, dry it in an electric blast drying oven at 55 °C and then weigh it again. Calculate the amount of extract, take it out and store it at -80 °C for standby.

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