Application of Chinese honeylocust fruit extract in killing trichomonad pigeon and preparation method of Chinese honeylocust fruit extract

Abstract

The invention discloses a Chinese honeylocust fruit extract for killing in-vitro cultured trichomonad pigeon and a preparation method of the Chinese honeylocust fruit extract, and belongs to the technical field of traditional Chinese medicines and the field of animal disease control, the Chinese honeylocust fruit extract is prepared by the following preparation method: crushing dry mature fruits of Chinese honeylocust fruits, performing condensation reflux extraction by using 70% ethanol, and collecting an extracting solution; concentrating by using a rotary evaporator; and drying at 55 DEG C by using an electrothermal blowing dry box to obtain the extract. According to the method, Chinese honeylocust fruit insecticidal components are extracted, and the method is easy to implement and convenient to operate. Compared with traditional chemical drugs, the alcohol extract prepared from medicinal saponin as a raw material has the advantages of no residue, no drug resistance, greenness and safety when being used for preventing and controlling trichomonad of pigeons, and has great popularization value. Meanwhile, the Chinese honeylocust fruit can effectively kill trichomonas pigeonae cultured in vitro, so that the adverse effect of trichomonas pigeonae on young pigeon growth is eliminated or reduced, and reference is provided for later application of the alcohol extract of the Chinese honeylocust fruit in trichomonas pigeonae resistance research and pigeon breeding.

Description

Technical Field

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

[0002] Trichomoniasis gallinae, also known as "pigeon canker" or "oral ulcer disease", is susceptible to pigeons of any breed, gender and age. In particular, squabs and young pigeons have a higher infection rate and incidence. After being infected with trichomonas, squabs show obvious clinical symptoms due to their weak disease resistance, and severely infected ones may die within 4 - 8 days. Trichomoniasis gallinae is a parasitic infectious disease that greatly harms the health of pigeons and restricts the sustainable development of the pigeon breeding industry. In the medical field, there have been many cases of using traditional Chinese medicine preparations to treat trichomonas infections. These preparations are extracted from natural plants, have a wide source, low toxicity and side effects, and also have various beneficial effects on the body of meat pigeons, gradually becoming a new research hotspot for the prevention and treatment of trichomoniasis gallinae. Therefore, it is necessary to explore a traditional Chinese medicine alternative drug with high efficiency and low toxicity against trichomonas gallinae. Summary of the Invention

[0003] The purpose of the present invention is to improve the existing extraction technology and develop a traditional Chinese medicine with high efficiency and low toxicity against trichomonas gallinae. The present invention provides a sophora japonica alcohol extract mainly made of sophora japonica and its preparation method, and studies the killing effect of the sophora japonica extract on trichomonas gallinae under normal in vitro culture conditions. The results show that a sophora japonica alcohol extract at a certain concentration can effectively kill trichomonas gallinae cultured in vitro, thereby reducing its adverse effects on the growth of young pigeons. The present invention provides a reference for the research on sophora japonica alcohol extract against trichomonas gallinae and its application in pigeon breeding.

[0004] To achieve the above purpose, the present invention provides a sophora japonica alcohol extract mainly made of sophora japonica. The sophora japonica extract mainly includes the following components: saponins, fatty acids, fisetin, flavokawain A, betulinic acid, betulin and friedelin.

[0005] Saponins, a natural surfactant, have excellent cleaning and foaming properties.

[0006] Triterpenoid saponins: have the effects of resolving phlegm and opening orifices, dissipating binds and detumescence, can stimulate the gastric mucosa to reflexively promote respiratory secretion to produce expectorant effects, and have inhibitory effects on Gram-negative enteric pathogenic bacteria and dermatophytes.

[0007] Tannins: have antibacterial and anti-inflammatory effects and can help treat some inflammatory diseases.

[0008] Ceryl alcohol: has certain antibacterial and anti-inflammatory effects.

[0009] Fatty acids: help regulate the body's fat metabolism.

[0010] Stigmasterol: It has antioxidant and anti-inflammatory effects.

[0011] These components of the sophora japonica extract jointly endow the sophora japonica extract with multiple effects, including antioxidant, anti-inflammatory, enhancing immune function, and promoting the absorption of tissue fat.

[0012] The present invention provides an ethanol extract of sophora japonica, which is mainly prepared from the following materials by weight: 10 g of sophora japonica fruit.

[0013] The preparation method of the above sophora japonica extract is as follows: First, crush the sophora japonica fruit with a pulverizer, pass through a 60-mesh sieve after crushing, weigh 5-10 g of sophora japonica powder in a clean round-bottom flask, 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 solution to the centrifuged precipitate again, carry out condensation reflux 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 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] As a further improvement of the present invention, the preparation method of the sophora japonica extract is as follows: First, crush the sophora japonica fruit with a pulverizer, pass through a 60-mesh sieve after crushing, weigh 10 g of sophora japonica powder in a clean round-bottom flask, 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 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 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 paste, take it out and store it at -80°C for standby.

[0015] Compared with the prior art, the beneficial effects of the present invention are:

[0016] 1. The insecticidal effect of the present invention is obvious. Through experiments, it is verified that the traditional Chinese medicine extract of the present invention has an obvious killing effect on Trichomonas gallinae cultured in vitro;

[0017] 2. As a pure Chinese herbal medicine, the present invention has the characteristics of natural raw materials, safety without residues, and no drug resistance. After medication, there will be no drug residues, which will not affect the quality of pigeon products and ensure the health of consumers; it is also not easy to cause drug resistance in Trichomonas gallinae and can be used stably for a long time;

[0018] 3. The preparation method of the sophora japonica extract provided by the present invention is simple, and the obtained extract has a high yield, effectively reducing the cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a graph showing the change in the mortality rate of Trichomonas gallinae cultured in vitro of the present invention over time under the action of sophora japonica extracts at different concentrations. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0020] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying 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.

[0021] Example 1

[0022] The present invention provides a sophora japonica extract, which is mainly prepared from the following materials by weight: 10 g of sophora japonica fruit and 30 times the amount of 70% ethanol solution.

[0023] For the preparation method of the sophora japonica extract in Example 1 of the present invention, first, the sophora japonica fruit is pulverized with a pulverizer, and after pulverization, it is sieved through a 60-mesh sieve. Take a clean round-bottom flask and weigh 10 g of sophora japonica powder, add 30 times the amount of 70% ethanol solution, let it stand for 30 min, reflux at 50 °C for 50 min, then centrifuge at 7500 r for 10 min to collect the supernatant. The precipitate obtained by centrifugation is continuously added with 30 times the amount of 70% ethanol solution, refluxed again for 30 min, and then the supernatant is collected. The collected supernatants are filtered by suction and the extract is concentrated into a paste with a rotary evaporator. Add 10 ml of distilled water and dissolve the paste by ultrasonic treatment, collect it with a pre-weighed centrifuge tube, dry it in an electric blast drying oven at 55 °C, weigh it again, calculate the amount of the extract paste, take it out and store it at -80 °C for standby.

[0024] Among them, the extract paste rate is: (weight of the extract paste after drying / weight of the crude drug) × 100%.

[0025] If the extract paste rate 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.

[0026] The sophora japonica extract paste rate is 31.00%.

[0027] Example 2

[0028] The present invention provides a sophora japonica extract, which is mainly prepared from the following materials by weight: 10 g of sophora japonica fruit and 30 times the amount of 60% ethanol solution.

[0029] The preparation method is the same as that of Example 1 except for the different ethanol concentrations.

[0030] Example 3

[0031] The present invention provides a sophora japonica extract, which is mainly prepared from the following materials by weight: 5 g of sophora japonica fruit and 30 times the amount of 60% ethanol solution.

[0032] The preparation method is the same as that of Example 1 except for the different raw material quality and ethanol concentration.

[0033] Example 4

[0034] The present invention provides a sophora japonica extract, which is mainly prepared from the following materials by weight: 5 g of sophora japonica fruit and 30 times the amount of 70% ethanol solution.

[0035] The preparation method is the same as that of Example 1 except for the different raw material quality.

[0036] Example 5

[0037] In order to clarify the effect of a sophora japonica extract in Example 1 of the present invention on killing Trichomonas gallinae in vitro culture, the sophora japonica extract in Example 1 was used as the experimental group for anti-Trichomonas gallinae experiment on Trichomonas gallinae.

[0038] The anti-Trichomonas gallinae experimental method of the sophora japonica extract includes the following steps:

[0039] S1. Trichomonas gallinae culture: The collected samples of diseased pigeons were put into HF complete medium and cultured in an incubator at 37°C. They were passaged with HF complete medium every 24 h. After stable culture for 3 generations or more, they were used for the experiment;

[0040] S2. Chinese medicine concentration ratio: The prepared sophora japonica crude drug concentration of 400 mg / mL was serially diluted with HF medium to prepare medicated media with 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;

[0041] Crude drug concentration: The mass / volume of the crude drug after conversion of the extraction rate;

[0042] S3. Anti-insect treatment: Prepare a density of 2.5×10 5A suspension of live worms at a concentration of [[[number]]] worms / mL was prepared. For the experimental groups, two replicates were set for each concentration. Four groups of metronidazole solutions (final concentration: 50 μg / mL) served as the positive control group, and four groups of double-distilled water served as the negative control group. In each tube, 120 μL of the worm suspension and 80 μL of the traditional Chinese medicine solution (80 μL of the positive control metronidazole solution and 80 μL of the negative control double-distilled water) were added, resulting in culture media containing the drug at final 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. The mixtures were thoroughly mixed and incubated at 37°C for 24 h.

[0043] S4. Effect observation: At 6 h, 12 h, 18 h, 24 h, and 48 h during the culture process, a 400-fold optical microscope was used to observe the killing of the worms, and the number of surviving trichomonads in each group was recorded. To ensure accurate results, for groups where all the worms had died, normal subculture (inoculating into antibiotic-free HF medium) was carried out, and the culture was continued for 24 h. If no live worms were determined, it could be judged that all the worms had been killed at that concentration.

[0044] Results and analysis

[0045] It can be seen from Table 1 that the killing effect of the sophorol extract reached 96% at 18 h when the final concentration of the crude drug was 10 mg / mL, and 100.0% at 24 h. From the perspective of the drug action time and effect observation, the complete killing effect on pigeon trichomonads can be achieved at 24 h.

[0046] Table 1 Observation of the killing effect of different drug concentrations

[0047]

[0048]

[0049] Note: - indicates that the number of well-growing worms is close to or exceeds the inoculation number.

[0050] In summary, it is shown that the killing 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 pigeon trichomonads. As a pure traditional Chinese medicine, it has the characteristics of natural raw materials, safety, no residue, and no drug resistance. After administration, there will be no drug residue, which does not affect the quality of pigeon products and ensures the health of consumers. It is also not easy for pigeon trichomonads to develop drug resistance, and it can be used stably for a long time.

[0051] It should be noted that in this text, 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 terms "comprising", "including" or any other variant thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device.

[0052] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

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

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

3. A preparation method of a sophora japonica extract, based on the sophora japonica extract described in claims 1-2, characterized in that, First, crush the Chinese honey locust fruit with a pulverizer, sieve it through a 60-mesh sieve after crushing, and store it at -80°C after crushing; weigh 5-10 g of Chinese honey locust powder in a clean round-bottom flask, add 30 times the amount of 60%-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 60%-70% ethanol solution to the centrifuged precipitate again, carry out condensation reflux 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 thermostatic 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 sophora japonica extract according to claim 3, characterized in that, First, crush the Chinese honey locust fruit with a pulverizer, sieve it through a 60-mesh sieve after crushing. Weigh 10 g of Chinese honey locust powder in a clean round-bottom flask, 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 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 thermostatic 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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