Preparation method of maggot extract and application of maggot extract in radiation damage resistance

Abstract

The invention provides a preparation method of a maggot extract and application of the maggot extract in radiation damage resistance, and the preparation method comprises the following steps: grinding dried maggot bodies into dry powder in a grinding machine, putting the dry powder into a Soxhlet extractor, digesting and refluxing for 1-4 hours by using ethanol, replacing the ethanol once every 1 hour in the refluxing process, cooling, and filtering dregs by using gauze to obtain the maggot extract. The preparation method comprises the following steps: uniformly spreading dregs in a flat plate, drying in a vacuum drying box, preparing a liquid medicine according to a ratio that 0.25-1L of double distilled water is added into every 50g of dried dregs, heating to boil by using a constant-temperature electric jacket, fully decocting for 2-6 hours, filtering, spreading the concentrated liquid medicine in a disc, drying in a freeze dryer, grinding into powder to obtain maggot extract powder, and storing in a refrigerator at the temperature of-20 DEG C for later use. The chrysomyia megacephala extract is obtained by adopting a water extraction method and has good biocompatibility and safety, and the chrysomyia megacephala extract has a good effect of relieving radiation-induced injury. The maggot extract has the advantages of being easy to obtain, efficient and small in toxic and side effect.

Description

Technical Field

[0001] The invention relates to the technical field of five-grain worm extracts, and in particular to a preparation method of the five-grain worm extract and application thereof in resisting radiation damage. Background Art

[0002] With the rapid development of my country's economy, radiation is widely used in various fields. While benefiting mankind, it also brings inevitable harm and poses a major threat to human health. In recent years, the number of radiation work units has gradually increased, and more and more working groups are susceptible to potential mild radiation damage, such as aerospace workers, scientific researchers, medical workers, etc.

[0003] Radiation not only causes significant damage to healthy tissues by destroying DNA and producing reactive oxygen species, but also causes imbalance of human immune function by producing immunostimulatory and immunosuppressive effects, and leads to cancer, physical deformity, and even death. In traditional Chinese medicine theory, radiation belongs to hot and toxic evil, which invades blood vessels, transmits to internal organs, damages qi and consumes yin, or causes dysfunction of related internal organs, imbalance of yin and yang in the body. Therefore, it is very necessary to develop a unique material basis of Chinese medicine to resist radiation damage to balance immune function. The drugs currently developed for resisting radiation damage are very limited, and there are also shortcomings such as strong side effects, high toxicity, and short half-life. Designing safe and efficient radiation protectors is a problem that needs to be solved urgently. Based on this hotspot, this application screens and studies many Chinese medicines, and finds that a five-grain worm extract has a good anti-radiation effect.

[0004] Grain worms are the larvae or pupae of Chrysomyia megacephala (Fabricius), an insect of the family Calliphoridae, and its closely related animals. They are cold and non-toxic. They taste sweet and salty, and enter the spleen meridian. They have the functions of strengthening the spleen and digesting food, removing heat and eliminating malnutrition. Modern studies have shown that the chemical components contained in the traditional Chinese medicine grain worms include proteins, fatty acids, chitin, etc., and it has been found that they have pharmacological effects such as antibacterial, promoting wound healing, lowering blood sugar and blood lipids, anti-tumor, anti-atherosclerosis, anti-inflammatory, immune regulation, angiogenesis, and tissue reconstruction. At present, there are no research reports on the application of grain worms in anti-radiation damage.

[0005] In view of the above research status, the purpose of this application is to provide a method for preparing a grain worm extract and its application in anti-radiation damage. This application first uses a water extraction method to prepare the grain worm extract, and then uses a freeze-drying method to dry it into powder; secondly, construct 60 A mouse model of mild radiation damage induced by Coγ-rays was developed. The extract of O. elegans was used as a therapeutic drug. The changes in mouse weight gain, fatigue resistance, peripheral blood cell count, organ index calculation, and expression levels of related immune factors were observed to evaluate the effectiveness of O. elegans extract in the treatment of radiation damage. Summary of the invention

[0006] In view of the limited number of safe and effective anti-radiation damage drugs, the present application provides a grain worm extract and a preparation method thereof and its application in anti-radiation damage. The present application firstly uses a water extraction method to prepare the grain worm extract, and then uses a freeze-drying method to dry it into powder; secondly, establishes 60 The mouse model of mild radiation damage caused by Coγ rays was treated with the extract of Ophiopogon japonicus, which increased the weight growth rate, anti-fatigue ability, peripheral blood cell level, organ index and immune-related factor level of mice, achieved the recovery effect of radiation-induced immune damage and functional decline, and had a good effect in resisting radiation damage.

[0007] The technical solution adopted by the present invention to solve the technical problem is:

[0008] A method for preparing a grain worm extract comprises the following steps:

[0009] Put the dried grain worm bodies into a grinder and grind them into dry powder, put them into a Soxhlet extractor, soak and reflux with ethanol for 1-4 hours, replace the alcohol every 1 hour during the reflux process, filter the residue with gauze after cooling, spread the residue on a flat plate, dry it in a vacuum drying oven, add 0.25-1L double distilled water for every 50g of dry residue to prepare the solution, heat it to boiling with a constant temperature electric heating mantle, fully boil it for 2-6 hours, spread the concentrated solution on a disc after filtering, put it into a freeze dryer for drying, and then crush it into powder to obtain the grain worm extract powder, which is stored in a -20℃ refrigerator for later use.

[0010] As a further technical solution of the present invention, the dried grain insect bodies are put into a grinder and ground into dry powder, and when put into a Soxhlet extractor, they are refluxed and boiled with ethanol for 1 hour; wherein, the medicinal solution is prepared at a ratio of 0.25L of double distilled water for every 50g of dry medicinal residue, and after being heated to boiling with a constant temperature electric heating mantle, it is fully decocted for 2 hours.

[0011] As a further technical solution of the present invention, the dried grain insect bodies are put into a grinder and ground into dry powder, and when put into a Soxhlet extractor, they are refluxed and boiled with ethanol for 2.5 hours; wherein, the medicinal solution is prepared at a ratio of 0.5L of double distilled water for every 50g of dry medicinal residue, and after being heated to boiling with a constant temperature electric heating mantle, it is fully decocted for 4 hours.

[0012] As a further technical solution of the present invention, the dried grain insect bodies are put into a grinder and ground into dry powder, and when put into a Soxhlet extractor, they are refluxed and boiled with ethanol for 4 hours; wherein, the medicinal solution is prepared at a ratio of 1L of double distilled water for every 50g of dry medicinal residue, and after being heated to boiling with a constant temperature electric heating mantle, it is fully decocted for 6 hours.

[0013] The use of the grain worm extract powder prepared by the above-mentioned grain worm extract preparation method in resisting radiation damage comprises the following steps:

[0014] 1) Animal grouping:

[0015] SPF female Kunming mice, 4 weeks old, weighing 25±4g, were randomly divided into five groups: blank control group, model group and three drug-treated groups. From the second day of radiation, the drug-treated groups were gavaged with O. quinquefolius extract every day, with a dose of 0.97mg / g and a single gavage volume of 0.4mL. The control group and model group mice were gavaged with an equal volume of normal saline.

[0016] 2) Irradiation parameters:

[0017] use 60 The mice were irradiated with Coγ rays at a single whole-body irradiation, with a distance of 0.933 m, a dose rate of 7304 mGy / h, an irradiation dose of 4 Gy, an irradiation time of 1972 s, and a projection area of ​​10 cm×10 cm. A weighted swimming experiment was performed on the 7th day after irradiation, and the body weight gain rate was measured on the 14th day. Peripheral blood tests were performed, organ indexes were calculated, and the levels of related immune factors were tested.

[0018] The beneficial effects of the present invention are:

[0019] The present application discloses a five grain worm extract and a preparation method thereof and its application in resisting radiation damage. Firstly, the five grain worm extract in the present application is derived from the natural Chinese medicine five grain worm, obtained by water extraction, and has good biocompatibility and safety.

[0020] Secondly, the five grain worm extract of the present application has a good effect on reducing radiation-induced damage, which refers to the damage caused by exposure of mice to 60 Coγ-ray exposure can cause slow weight gain, decreased physical strength, tissue inflammation, and immune damage.

[0021] In addition, the five-grain worm extract of the present application can be made into anti-radiation products such as food, medicine or health care products because of its easy acquisition, high efficiency and small toxic side effects. It has good application prospects and high social and economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a flow chart of the experiment on the anti-radiation damage of the five-grain insect extract in the embodiment of the present invention.

[0023] Figure 2 It is the weight growth rate of each group of mice in the embodiment of the present invention.

[0024] Figure 3 It is the weight-loaded exhaustive swimming time of each group of mice in the examples of the present invention.

[0025] Figure 4 Comparison of the number of white blood cells in the blood of each group of mice in the examples of the present invention.

[0026] Figure 5 The figure is a comparison of the number of lymphocytes in the blood of each group of mice in the examples of the present invention.

[0027] Figure 6 The figure is a comparison of the number of neutrophils in the blood of each group of mice in the examples of the present invention.

[0028] Figure 7 This is a comparison of the thymus index of each group of mice in the examples of the present invention.

[0029] Figure 8 1 is a comparison of the spleen index of each group of mice in the examples of the present invention.

[0030] Fig. 9 This is a comparison of the serum IGFBP-3 factor levels of each group of mice in the examples of the present invention. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] The embodiment of the present invention provides a method for preparing a grain worm extract, comprising the following steps:

[0033] Put the dried grain worm bodies into a grinder and grind them into dry powder, put them into a Soxhlet extractor, and soak and reflux with 95% ethanol for 1 to 4 hours. During the reflux process, the alcohol is replaced every 1 hour. After cooling, the residue is filtered with gauze, and the residue is spread evenly on a flat plate, and dried in a 60°C vacuum drying oven. The liquid medicine is prepared by adding 0.25 to 1L of double distilled water for every 50g of dry residue. The constant temperature electric heating mantle is heated to boiling, and the medicine is fully decocted for 2 to 6h. After filtering, the concentrated liquid medicine is spread on a disc, put into a freeze dryer and dry for 3 days, and crushed into powder to obtain the grain worm extract powder. It is stored in a -20°C refrigerator for use. When using, the freeze-dried powder is added to double distilled water, and heated and shaken with an ultrasonic oscillator for 30 minutes to fully dissolve it.

[0034] Determination of protein content in grain worm extract:

[0035] The protein content of the grain worm extract was determined by BCA method. 30 μL of 5 mg / mL BSA standard solution was added to 120 μL PBS solution to prepare a BSA protein standard with a final concentration of 1 mg / mL. The 1 mg / mL standard was diluted to 0.75, 0.5, 0.25, 0.125, 0.025 and 0 mg / mL in proportion; an appropriate amount of grain worm extract powder was weighed and dissolved in double distilled water to prepare a grain worm extract sample with a concentration of 1 mg / mL; 1 mL of reagent A was added to 20 μL of reagent B to prepare the BCA working solution. According to the instructions of the kit, 2 μL of BSA protein standards and grain worm extract samples of each concentration were added to a 96-well plate, diluted to 20 μL with PBS solution, and then 200 μL of BCA working solution was added. Protect from light and incubate at 37 ° C for 30 minutes. Take out the well plate and put it into the microplate reader to measure the absorbance at 562nm. Set the horizontal axis to the concentration of each BSA standard, and the vertical axis to the absorbance value of each sample at 562nm. Draw a standard curve and calculate the protein content of each sample. The obtained concentration is the concentration of each sample diluted 10 times.

[0036] The use of the grain worm extract powder prepared by the above-mentioned grain worm extract preparation method in resisting radiation damage comprises the following steps:

[0037] 1) Animal grouping:

[0038] SPF Kunming (KM) female mice, 4 weeks old, weighing 25±4g, were randomly divided into five groups (n=8): blank control group, model group, and treatment group (Ceratitis 1 group, Ceratitis 2 group, and Ceratitis 3 group). From the second day of radiation, the treatment groups were gavaged with Ceratitis extract every day, with a dose of 0.97mg / g and a single gavage volume of 0.4mL. The control group and model group mice were gavaged with an equal volume of normal saline.

[0039] 2) Irradiation parameters:

[0040] use 60 The mice were irradiated with Coγ rays at a single whole-body irradiation, with a distance of 0.933 m, a dose rate of 7304 mGy / h, an irradiation dose of 4 Gy, an irradiation time of 1972 s, and a projection area of ​​10 cm×10 cm. A weighted swimming experiment was performed on the 7th day after irradiation, and the body weight gain rate was measured on the 14th day. Peripheral blood tests were performed, organ indexes were calculated, and the levels of related immune factors were tested.

[0041] 3) Experimental content:

[0042] 3.1) Changes in weight gain

[0043] The efficacy of the grain worms obtained by different preparation processes was evaluated to screen the optimal process parameters. The mice were weighed on the 2nd day after irradiation and weighed again on the 14th day to calculate the weight growth rate. Weight growth rate = (weight of mice on the 14th day - weight of mice on the 1st day) / weight of mice on the 1st day * 100%.

[0044] 3.2) Weighted swimming test

[0045] On the 7th day after radiation, 0.5 h after gavage, a wire weighing 5.0% of the mouse's body weight was placed 1 cm from the base of the mouse's tail, and the mouse's fur was sprayed with a spray bottle. The mouse was allowed to move freely in a water tank at a depth of 50 cm and a temperature of 28-30°C until exhaustion, and the time was counted. The exhaustion time was the interval from the time the mouse was placed in the water to the time it sank into the water and did not float up again, which was 8 seconds.

[0046] 3.3) Routine blood test

[0047] On the 14th day after irradiation, the mice in each group were weighed, their eyeballs were removed and blood was collected. The blood was placed in a 1.5 mL centrifuge tube containing EDTA, and the number of white blood cells (WBC), neutrophils (NE), and lymphocytes (LY) in the whole blood of the mice in each group was detected using a hematology analyzer.

[0048] 3.4) Organ index

[0049] On the 14th day after radiation, the mice were killed by dislocating the cervical vertebrae, the thymus and spleen were separated, the fat was removed, the blood was washed off with pre-cooled saline, the surface moisture of the organs was absorbed with filter paper, and the weight was weighed to calculate the organ index of the mice. Organ index = organ weight / body weight.

[0050] 3.5) Levels of immune-related factors

[0051] 3.5.1) Serum IGFBP-3 level

[0052] On the 14th day after irradiation, the mice in each group were weighed, and the eyeballs were removed to collect about 0.5 mL of blood in a 1.5 mL centrifuge tube. The blood was allowed to stand at room temperature for 1 hour, and then centrifuged at 1000xg, 4°C, 10 minutes to separate the serum in time. The IGFBP-3 factor level in mouse serum was detected according to the instructions of the ELISA kit.

[0053] Example 1: Preparation and content determination of five grain insect extract 1

[0054] Put the dried grain insect body into a grinder and grind it into dry powder, put it into a Soxhlet extractor, and reflux it with 95% ethanol for 1 hour. Change the alcohol every 1 hour during the reflux process. After cooling, filter the drug residue with gauze, spread the drug residue on a flat plate, and dry it in a 60℃ vacuum drying oven. Prepare the drug solution by adding 0.25L double distilled water to every 50g of dry drug residue, heat it to boiling with a constant temperature electric heating jacket, and fully decoct it for 2 hours. After filtering, spread the concentrated drug solution on a disc, put it in a freeze dryer and dry it for 3 days, crush it into powder, and store it in a -20℃ refrigerator for standby use. When using, add the freeze-dried powder to an appropriate amount of double distilled water, heat and shake it with an ultrasonic oscillator for 30 minutes to fully dissolve it, and administer 1 group of grain insects.

[0055] According to the BCA test results, the protein content in the grain worm extract sample with a concentration of 1 mg / mL was 50.34 μg / mL

[0056] Example 2: Preparation and content determination of five grain insect extract 2

[0057] Put the dried grain insect body into a grinder and grind it into dry powder, put it into a Soxhlet extractor, and reflux it with 95% ethanol for 2.5 hours. Change the alcohol every 1 hour during the reflux process. After cooling, filter the drug residue with gauze, spread the drug residue on a flat plate, and dry it in a 60℃ vacuum drying oven. Prepare the drug solution by adding 0.5L double distilled water to every 50g of dry drug residue, heat it to boiling with a constant temperature electric heating jacket, and fully decoct it for 4 hours. After filtering, spread the concentrated drug solution on a disc, put it in a freeze dryer and dry it for 3 days, crush it into powder, and store it in a -20℃ refrigerator for standby use. When using, add the freeze-dried powder to an appropriate amount of double distilled water, heat and shake it with an ultrasonic oscillator for 30 minutes to fully dissolve it, and administer it to 2 groups of grain insects.

[0058] According to the BCA test results, the protein content in the grain worm extract sample with a concentration of 1 mg / mL was 62.46 μg / mL.

[0059] Example 3: Preparation and content determination of five grain insect extract 3

[0060] Put the dried grain insect body into a grinder and grind it into dry powder, put it into a Soxhlet extractor, and reflux it with 95% ethanol for 4 hours. Change the alcohol every 1 hour during the reflux process. After cooling, filter the residue with gauze, spread the residue on a flat plate, and dry it in a 60℃ vacuum drying oven. Prepare the medicine solution by adding 1L of double distilled water to every 50g of dry residue, heat it to boiling with a constant temperature electric heating jacket, and fully decoct it for 6 hours. After filtering, spread the concentrated medicine solution on a disc, put it in a freeze dryer and dry it for 3 days, crush it into powder, and store it in a -20℃ refrigerator for standby use. When using, add the freeze-dried powder to an appropriate amount of double distilled water, heat and shake it with an ultrasonic oscillator for 30 minutes to fully dissolve it, and administer it to 3 groups of grain insects.

[0061] According to the BCA test results, the protein content in the five-grain worm extract sample with a concentration of 1 mg / mL was 79.79 μg / mL.

[0062] Example 4: Effects of three groups of five-grain insect extracts on weight changes in mice

[0063] Four-week-old SPF female Kunming (KM) mice weighing 25±4 g were randomly divided into five groups (n=8): control group, model group, and drug-treated group (grain worm group 1, grain worm group 2, and grain worm group 3). 60 The mice in the model group and each treatment group were irradiated with Coγ rays once, with a dose of 4Gy. From the second day of radiation, the three treatment groups were gavaged with three kinds of grain worm extracts prepared by three preparation processes every day, with a dose of 0.97 mg / g and a single gavage volume of 0.4 mL. The mice in the control group and the model group were gavaged with an equal volume of normal saline. The body weight of each group of mice was measured on the second and fourteenth days after radiation, and the body weight growth rate was calculated. Body weight growth rate = (weight of mice on the 14th day - weight of mice on the first day) / weight of mice on the first day * 100%. The results are shown in the attached figure. Figure 2 shown.

[0064] As the instruction manual Figure 2 The weight growth rate of the model group was lower, and the weight growth rates of the three drug-administered groups were significantly higher than those of the model group (P<0.01; P<0.001; P<0.0001), among which the weight growth rate of the grain worm group 3 was significantly higher than that of the grain worm group 2 and grain worm group 3 (P<0.01; P<0.0001). The above results show that radiation can slow down the weight growth of mice. The grain worm extract prepared in this application can effectively avoid the effect of irradiation on the slow weight growth of mice, and this effect can be significantly improved by prolonging the decoction time.

[0065] Combining this example with the results of protein content detection by the BCA kit, subsequent experiments adopted the conditions in Example 3 to prepare the Osmanthus fragrans extract for administration.

[0066] Example 5: Effect on the anti-fatigue ability of mice

[0067] On the 7th day after radiation, 0.5h after gavage, a wire weighing 5.0% of the body weight was placed 1cm from the base of the tail of each group of mice. The mice were sprayed with a spray bottle to wet their fur. They were allowed to move freely in a water tank with a depth of 50cm and a temperature of 28-30℃ until they were exhausted and the time was counted. The time to exhaustion was the interval from the time they were placed in the water to the time they sank into the water and no longer floated up for 8s. The results are shown in the attached Figure 3 shown.

[0068] As the instruction manual Figure 3The weight-exhaustive swimming time of the model group was significantly shorter than that of the control group, and the difference was significant (P<0.05); the weight-exhaustive swimming time of the drug-treated group was significantly longer than that of the model group (P<0.05). The results show that irradiation leads to excessive production of free radicals, which eventually leads to oxidative damage to the cell membrane and fatigue of the body. The extract of Osmanthus fragrans has a significant promoting effect on the physical recovery of mice after irradiation, indicating that the extract has a strong antioxidant capacity, can effectively inhibit the production of free radicals, and has a significant repair effect on cell and mitochondrial damage caused by free radicals, thereby delaying fatigue.

[0069] Example 6: Effects on peripheral blood cell levels in mice

[0070] Radiation can have adverse effects on the hematopoietic system, accompanied by varying degrees of leukopenia. When the body tissue is damaged, inflammation occurs, and peripheral blood lymphocytes are also severely damaged, with a sharp decrease in number. The purpose of this example is to detect the number of white blood cells (WBC), lymphocytes (LY) and neutrophils (NE) in the blood of mice to observe the effect of the grain worm extract on the reduction of blood immune function in mice caused by radiation.

[0071] On the 14th day after irradiation, the mice in each group were weighed, their eyeballs were removed and blood was collected. The blood was placed in a 1.5 mL centrifuge tube containing EDTA. The number of white blood cells (WBC), lymphocytes (LY) and neutrophils (NE) in the whole blood of the mice in each group was measured using a hematology analyzer. The results are shown in the attached figure. Figure 4 , Figure 5 , Figure 6 As shown, the levels of WBC, LY and NE in the blood of irradiated mice without medication decreased, while the medication group showed the opposite trend and the differences were significant (P<0.01; P<0.0001; P<0.0001), indicating that γ rays caused a decrease in blood WBC, LY and NE, thereby reducing the blood immune function of mice. The five-grain worm extract prepared in this application can reverse the imbalance of immune cell levels caused by this radiation, thereby promoting the recovery of blood immune function.

[0072] Example 7: Effect on mouse thymus index

[0073] Irradiation usually causes damage to the organs of the body, causing its weight to change accordingly. As one of the most important immune organs of the body, the thymus gland has the main function of cultivating various T lymphocytes, and regulating immune function and differentiating mature T cells by secreting thymus hormones and cytokines through stromal cells. Therefore, the weight of the thymus gland can reflect the quality of the overall immune function to a certain extent. The present embodiment aims to observe the influence of the five grain insect extract on the decline of the central immune function of mice caused by radiation by calculating the thymus gland index.

[0074] On the 14th day after radiation, the mice were killed by dislocating the cervical vertebrae, the thymus was separated, the fat was removed, the blood was washed off with pre-cooled saline, the surface moisture of the organ was absorbed with filter paper, and the weight was weighed to calculate the organ index of the mouse. Thymus index = thymus weight / body weight. The results are shown in the attached figure. Figure 7 As shown, the thymus index of the model group was significantly lower than that of the control group and the drug-treated group (P<0.001; P<0.01), which indicates that radiation damages the immune organs, resulting in reduced immunity. For the damage to the immune organs caused by irradiation, the grain worm extract prepared in this application can significantly reverse the damage to the thymus and improve the immune function of mice.

[0075] Example 8: Effect on the spleen index of mice

[0076] The spleen is mainly composed of B lymphocytes, T lymphocytes, macrophages, etc. It is the main site of immune response and an important base for antigens to produce immune effector substances. A decrease in the spleen index will affect humoral immunity and cellular immunity, which will lead to a decrease in immunity. The spleen index reflects the differences in immune function of mice in different groups. The purpose of this example is to calculate the spleen index to observe the effect of the grain worm extract on the decline in peripheral immune function of mice induced by radiation.

[0077] On the 14th day after radiation, mice were killed by dislocating the cervical vertebrae, the spleen was separated, fat was removed, blood stains were washed off with pre-cooled saline, the surface moisture of the organ was absorbed with filter paper, and the weight was calculated. Spleen index = spleen weight / body weight. The results are shown in the attached figure. Figure 8 As shown, the spleen index of the model group was significantly lower than that of the control group and the drug-treated group (P<0.05), which indicates that radiation damages the peripheral immune system, resulting in reduced immunity. For spleen damage caused by irradiation, the grain worm extract prepared in this application has a reversal effect on spleen damage caused by gamma rays, indicating that grain worms can improve the immune function of mice to a certain extent.

[0078] Example 9: Effect on the level of IGFBP-3 factor in mouse serum

[0079] IGFBP-3 is a growth inhibitor induced by p53 and is radiosensitive. p53 is an important component of the DNA damage repair pathway. Under the stimulation of DNA damage induced by factors such as ionizing radiation, the upregulation of IGFBP-3 dependent on p53 is considered to be part of the DNA damage response. This example aims to observe the effect of the grain worm extract on radiation-induced DNA damage by measuring the level of IGFBP-3 factor in mouse serum.

[0080] On the 14th day after irradiation, the mice in each group were weighed, their eyeballs were removed, and about 0.5 mL of blood was collected in a 1.5 mL centrifuge tube. The blood was allowed to stand at room temperature for 1 hour, and then centrifuged at 1000 x g, 4°C, for 10 minutes. The serum was separated in time. The IGFBP-3 factor level in the mouse serum was detected according to the instructions of the ELISA kit. The results are shown in the attached figure. Fig. 9 The IGFBP-3 level in the model group was significantly higher than that in the control group and the drug-treated group (P<0.05; P<0.01), indicating that radiation increases the IGFBP-3 level, which may cause DNA damage. The grain worm extract prepared in this application can significantly reduce the IGFBP-3 level after radiation, treat DNA damage, and play an anti-radiation role.

[0081] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0082] The above are only preferred embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for preparing a grain worm extract, characterized in that: The steps include: Put the dried grain worm bodies into a grinder and grind them into dry powder, put them into a Soxhlet extractor, soak and reflux with ethanol for 1-4 hours, replace the alcohol every 1 hour during the reflux process, filter the residue with gauze after cooling, spread the residue on a flat plate, dry it in a vacuum drying oven, add 0.25-1L double distilled water for every 50g of dry residue to prepare the solution, heat it to boiling with a constant temperature electric heating mantle, fully boil it for 2-6 hours, spread the concentrated solution on a disc after filtering, put it into a freeze dryer for drying, and then crush it into powder to obtain the grain worm extract powder, which is stored in a -20℃ refrigerator for later use.

2. The method for preparing a grain worm extract according to claim 1, characterized in that: The dried grain insect bodies are put into a grinder and ground into dry powder, and then put into a Soxhlet extractor and refluxed and boiled with ethanol for 1 hour; wherein, the medicine solution is prepared by adding 0.25L double distilled water for every 50g of dry medicine residue, and the medicine solution is heated to boiling with a constant temperature electric heating mantle and fully boiled for 2 hours.

3. The method for preparing a grain worm extract according to claim 1, characterized in that: The dried grain insect bodies are put into a grinder and ground into dry powder, and then put into a Soxhlet extractor and refluxed and boiled with ethanol for 2.5 hours; wherein, the medicine solution is prepared by adding 0.5L of double distilled water for every 50g of dry medicinal residue, and the medicine solution is heated to boiling with a constant temperature electric heating mantle and fully boiled for 4 hours.

4. The method for preparing a grain worm extract according to claim 1, characterized in that: The dried grain insect bodies are put into a grinder and ground into dry powder, and then put into a Soxhlet extractor and refluxed and boiled with ethanol for 4 hours; wherein, the medicine solution is prepared at a ratio of 1L of double distilled water per 50g of dry medicine residue, and the medicine solution is heated to boiling with a constant temperature electric heating mantle and fully boiled for 6 hours.

5. Use of the grain worm extract powder prepared by the method for preparing the grain worm extract according to any one of claims 1 to 4 in resisting radiation damage, characterized in that: The following steps are involved: 1) Animal grouping: SPF female Kunming mice, 4 weeks old, weighing 25±4g, were randomly divided into five groups: blank control group, model group, and treatment group. From the second day of radiation, the treatment group was gavaged with Ophiopogon japonicus extract every day, with a dose of 0.97mg / g and a single gavage volume of 0.4mL. The control group and model group mice were gavaged with an equal volume of normal saline. 2) Irradiation parameters: use 60 The mice were irradiated with Coγ rays at a single whole-body irradiation, with a distance of 0.933 m, a dose rate of 7304 mGy / h, an irradiation dose of 4 Gy, an irradiation time of 1972 s, and a projection area of ​​10 cm×10 cm. A weighted swimming experiment was performed on the 7th day after irradiation, and the body weight gain rate was measured on the 14th day. Peripheral blood tests were performed, organ indexes were calculated, and the levels of related immune factors were tested.

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