Traditional Chinese medicine composition and application thereof in preparation of radiation damage protection products

Abstract

The invention discloses a traditional Chinese medicine composition and application thereof in preparation of radiation damage protection products. The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 3-6 parts of fructus lycii, 1 part of radix ginseng, 1-3 parts of fructus crataegi and 1-3 parts of green tea. The traditional Chinese medicine composition provided by the invention is prepared from natural medicinal and edible raw materials, has the effect of preventing and treating radiation damage, can be taken for a long time, and has no toxic or side effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine, and particularly relates to a traditional Chinese medicine composition and its application in the preparation of products for protecting against radiation damage. Background Art

[0002] In modern society, from the wide application of nuclear energy technology to tumor radiotherapy in clinical medicine, from the radio waves of mobile phones and computers to the daily sunlight ultraviolet rays, radiation has become an existence that cannot be ignored. While bringing convenience to people, radiation also has a certain impact on health. Some studies have shown that radiation can generate reactive oxygen species (ROS) free radicals, interfere with macromolecules such as DNA and proteins, and in severe cases, can cause cell damage and functional abnormalities, and even lead to functional disorders, lesions, and even death of multiple organs of the body.

[0003] There are some components with anti-radiation effects in traditional Chinese medicine and natural medicines, which can be used as potential anti-radiation drugs. A large number of experiments have confirmed that traditional Chinese medicine has good anti-radiation effects. The traditional Chinese medicine "Anduolin Capsule" is the first approved anti-radiation traditional Chinese medicine in China, and Angelica sinensis, Eleutherococcus senticosus, and Rhodiola rosea also have good anti-radiation effects. Compared with similar chemical drugs, traditional Chinese medicine and natural medicines have the characteristics of multi-component and multi-target effects, and have many advantages such as long action time, low cost, and no obvious toxic and side effects. In view of this, the present invention is specifically proposed. Summary of the Invention

[0004] The object of the present invention is to provide a traditional Chinese medicine composition prepared from natural raw materials for both medicine and food, its preparation method and application. The traditional Chinese medicine composition has the function of preventing and treating radiation damage, can be taken for a long time, and has no toxic and side effects.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] In a first aspect, the present invention provides a traditional Chinese medicine composition, which is prepared by extracting raw materials including the following parts by weight: 3-6 parts by weight of wolfberry fruits, 1 part by weight of ginseng, 1-3 parts by weight of hawthorn fruits, and 1-3 parts by weight of green tea.

[0007] Preferably, the traditional Chinese medicine composition is prepared by extracting raw materials including the following parts by weight: 4.5-6.5 parts by weight of wolfberry fruits, 1 part by weight of ginseng, 2-3.5 parts by weight of hawthorn fruits, and 2-3 parts by weight of green tea.

[0008] According to an embodiment of the present invention, the traditional Chinese medicine composition is prepared by extracting raw materials including the following parts by weight: 10 parts by weight of wolfberry fruits, 3 parts by weight of ginseng, 3 parts by weight of hawthorn fruits, and 5 parts by weight of green tea.

[0009] According to an embodiment of the present invention, the traditional Chinese medicine composition is prepared by extracting raw materials including the following parts by weight: 8 parts by weight of wolfberry fruits, 2 parts by weight of ginseng, 4 parts by weight of hawthorn fruits, and 4 parts by weight of green tea.

[0010] According to an embodiment of the present invention, the traditional Chinese medicine composition is prepared by extracting raw materials including the following parts by weight: 10 parts by weight of wolfberry fruits, 2 parts by weight of ginseng, 5 parts by weight of hawthorn fruits, and 3 parts by weight of green tea.

[0011] In a second aspect, the present invention further provides a preparation method of the traditional Chinese medicine composition, including the following steps: subjecting each raw material to alcohol extraction or water extraction to obtain the composition.

[0012] The reagent used for alcohol extraction is an ethanol aqueous solution with a volume concentration not exceeding 50%. Considering the actual application situation, an ethanol aqueous solution with a volume concentration not exceeding 30% is preferred, such as a 20-30% ethanol aqueous solution.

[0013] The method of alcohol extraction is reflux extraction or percolation extraction.

[0014] The conditions for alcohol extraction are as follows: the number of extraction times is 1-3 times, preferably 3 times; the extraction time is 1-2 hours; and each extraction is carried out according to the ratio of using 6-10 mL of alcohol reagent per 1 g of raw material.

[0015] The method of water extraction is decoction extraction or reflux extraction.

[0016] The conditions for water extraction are as follows: the number of extraction times is 1-3 times; the extraction time is 1-2 hours; and each extraction is carried out according to the ratio of using 6-12 mL of water per 1 g of raw material.

[0017] According to an embodiment of the present invention, the traditional Chinese medicine composition can be prepared according to the following two extraction methods:

[0018] The first extraction method: mixing each raw material and then carrying out alcohol extraction or water extraction, concentrating the filtrate, performing alcohol precipitation, filtering / centrifuging to obtain a filtrate; concentrating the filtrate, drying to obtain the traditional Chinese medicine composition;

[0019] The second extraction method: subjecting each raw material to water extraction separately, concentrating the filtrate, performing alcohol precipitation, filtering / centrifuging to obtain each filtrate; concentrating each filtrate separately, drying, pulverizing, and mixing to obtain the traditional Chinese medicine composition.

[0020] The reagent used for alcohol precipitation is absolute ethanol or an ethanol aqueous solution with a volume concentration of 95%; considering the actual application situation, an ethanol aqueous solution with a volume concentration of 95% is preferred.

[0021] The drying method includes but is not limited to methods such as vacuum drying under reduced pressure and freeze drying.

[0022] The concentration is increased to 0.5 - 1.2 g of crude drug / mL, preferably 0.6 - 1.0 g of crude drug / mL, such as 0.8 g of crude drug / mL or 1.0 g of crude drug / mL.

[0023] Exemplarily, the raw material is wolfberry fruit. Extraction is carried out at a ratio of 6 - 10 mL of water added per 1 g of wolfberry fruit. The extract is filtered and concentrated to 0.8 g of crude drug / mL. Ethanol is added to the concentrated solution until the volume concentration is 60%. After filtration / centrifugation, the filtrate is concentrated and dried to obtain wolfberry extract.

[0024] Exemplarily, the raw material is ginseng. Extraction is carried out at a ratio of 6 - 10 mL of water added per 1 g of ginseng. The extract is filtered and concentrated to 1.0 g of crude drug / mL. Ethanol is added to the concentrated solution until the volume concentration is 50%. After filtration / centrifugation, the filtrate is concentrated and dried to obtain ginseng extract.

[0025] Exemplarily, the raw material is hawthorn. The hawthorn is crushed. Extraction is carried out at a ratio of 6 - 10 mL of water added per 1 g of hawthorn. The extract is filtered and concentrated to 0.5 g of crude drug / mL. Ethanol is added to the concentrated solution until the volume concentration is 30%. After filtration / centrifugation, the filtrate is concentrated and dried to obtain hawthorn extract.

[0026] Exemplarily, the raw material is green tea. The green tea is crushed. Extraction is carried out at a ratio of 8 - 12 mL of water added per 1 g of green tea. The extract is filtered and concentrated to 0.9 g of crude drug / mL. Ethanol is added to the concentrated solution until the volume concentration is 50%. After filtration / centrifugation, the filtrate is concentrated and dried to obtain green tea extract.

[0027] According to a preferred embodiment of the present invention, the traditional Chinese medicine composition is prepared according to the following steps:

[0028] After taking the raw material mixture in proportion, extraction is carried out at a ratio of 6 - 10 mL of water added per 1 g of raw material. The extract is filtered and concentrated to 0.8 g of crude drug / mL. Ethanol is added to the concentrated solution until the concentration is 60%. After filtration / centrifugation, the filtrate is concentrated and dried to obtain the traditional Chinese medicine composition;

[0029] According to a preferred embodiment of the present invention, the traditional Chinese medicine composition is prepared according to the following steps:

[0030] The raw materials, Chinese wolfberry, ginseng, hawthorn, and green tea, are decocted according to the ratio of adding 6 - 12 mL of water to every 1 gram of raw material respectively. The extracts are filtered and concentrated to 0.8 g of crude drug / mL, 1.0 g of crude drug / mL, 0.5 g of crude drug / mL, and 0.9 g of crude drug / mL respectively. Ethanol is added to each concentrated solution to a concentration of 60%, 50%, 30%, and 50% respectively, and then filtered / centrifuged. The filtrates are concentrated and dried to obtain the extracts; the extracts are pulverized, sieved, and mixed to obtain the traditional Chinese medicine composition.

[0031] The drying method includes but is not limited to methods such as vacuum drying under reduced pressure and freeze drying.

[0032] In a third aspect, the present invention further provides a traditional Chinese medicine composition, which is composed of the following components in parts by weight: 4.5 - 6.5 parts by weight of wolfberry extract, 1 part by weight of ginseng extract, 2 - 3.5 parts by weight of hawthorn extract, 2 - 3 parts by weight of green tea extract, and 12.5 - 15 parts by weight of inulin.

[0033] In a fourth aspect, the present invention further provides a traditional Chinese medicine preparation, which includes the traditional Chinese medicine composition obtained by the above extraction or a traditional Chinese medicine composition formed by adding inulin on the basis of the traditional Chinese medicine composition obtained by the extraction.

[0034] The dosage form of the traditional Chinese medicine preparation is powder, granule, oral liquid, capsule, tablet, or effervescent tablet.

[0035] The traditional Chinese medicine preparation includes a single - ingredient dosage form or a compound dosage form.

[0036] In a fifth aspect, the present invention further provides the application of the above - mentioned traditional Chinese medicine composition and traditional Chinese medicine preparation in the preparation of products for preventing or treating radiation injury protection.

[0037] The products include food, health products, or pharmaceutical preparations;

[0038] The radiation injury includes one or more of the injuries caused by alpha rays, beta rays, gamma rays, X - rays, ultraviolet rays, microwaves, and radio waves.

[0039] The beneficial effects obtained by the present invention are as follows:

[0040] 1. The traditional Chinese medicine composition provided by the present invention is prepared from natural raw materials that can be used both as medicine and food. It has an obvious protective effect on the damage models of Caenorhabditis elegans and mice irradiated by gamma rays and X - rays, and can be made into radiation injury protection products such as natural radiation protectants for preventing or treating radiation - induced related diseases and symptoms. It has no toxic and side effects and can be taken for a long time.

[0041] 2. On the basis of the above traditional Chinese medicine composition, the present invention adds inulin, which can not only be used as an auxiliary material in the preparation, but also act as an active ingredient to synergistically act with wolfberry extract, ginseng extract, hawthorn extract, and green tea extract to further improve the anti-radiation ability. Description of the Drawings

[0042] Figure 1 For the number of eggs laid by N2 Caenorhabditis elegans with radiation damage caused by 60 Co-γ rays; in the figure: * indicates compared with Control, * indicates P value less than 0.05, ** indicates P value less than 0.01, *** indicates P value less than 0.001, **** indicates P less than 0.0001, # indicates compared with the 20 Gy group, # P value less than 0.05, ## indicates P value less than 0.01, indicates P value less than 0.001, # indicates P less than 0.0001; ns: no significant difference.

[0043] Figure 2 For the effect of FHGL on 60 the survival rate of N2 Caenorhabditis elegans with radiation damage caused by Co-γ rays.

[0044] Figure 3 For the effect of FHGL on the number of eggs laid by Caenorhabditis elegans with radiation damage caused by X-rays; in the figure: * indicates compared with Control, * indicates P value less than 0.05, ** indicates P value less than 0.01, *** indicates P value less than 0.001, **** indicates P less than 0.0001, # indicates compared with the 20 Gy group, # P value less than 0.05, ## indicates P value less than 0.01, indicates P value less than 0.001, # indicates P less than 0.0001; ns: no significant difference.

[0045] Figure 4 For the effect of FHGL on the body weight of C57BL / 6 mice after irradiation with 6.0 Gy γ-rays.

[0046] Figure 5 For the effect of FHGL on LY% of C57BL / 6 mice after radiation; in the figure: compared with the normal control group, *p < 0.05, **p < 0.01, ***p < 0.001; compared with the model group, # p < 0.05, ## p < 0.01, ### p < 0.001.

[0047] Figure 6 For the effect of FHGL on NE% of C57BL / 6 mice after radiation; in the figure: compared with the normal control group, *p < 0.05, **p < 0.01, ***p < 0.001; compared with the model group, #p < 0.05, ## p < 0.01, ### p < 0.001.

[0048] Figure 7 Effect of FHGL on MO% in C57BL / 6 mice after radiation; In the figure: compared with the normal control group, ***p < 0.001; compared with the total body irradiation group, # p < 0.05. Detailed implementation mode

[0049] The present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments.

[0050] The experimental methods used in the following embodiments are all conventional methods unless otherwise specified.

[0051] The reagents, materials, instruments, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0052] Example 1. Preparation of traditional Chinese medicine composition

[0053] Formula: 10 kg of wolfberry fruit, 3 kg of ginseng, 3 kg of hawthorn, 5 kg of green tea.

[0054] Preparation method: Crush 21 kg of raw materials including 10 kg of wolfberry fruit, 3 kg of ginseng, 3 kg of hawthorn, and 5 kg of green tea into coarse powder, soak in water for 1 hour, extract by heating and boiling with 8 times the weight of water for a total of 3 times, 2 hours each time, concentrate to 0.8 g of crude drug / ml, add ethanol to a concentration of 50%, filter / centrifuge, concentrate the filtrate, and dry under reduced pressure in vacuum to obtain 4.6 kg of extract.

[0055] Example 2. Preparation of traditional Chinese medicine composition

[0056] Formula: 2.0 kg of wolfberry fruit, 0.5 kg of ginseng, 1.0 kg of hawthorn, 1.0 kg of green tea.

[0057] Preparation method: Crush 4.5 kg of raw materials including 2.0 kg of wolfberry fruit, 0.5 kg of ginseng, 1.0 kg of hawthorn, and 1.0 kg of green tea into coarse powder, soak in 30% ethanol for 0.5 hour, percolate with 30 times the weight of 30% ethanol, concentrate, and dry under reduced pressure in vacuum to obtain 0.95 kg of extract.

[0058] Example 3. Preparation of traditional Chinese medicine composition

[0059] Formula: 10.0 kg of wolfberry fruit, 2.0 kg of ginseng, 5.0 kg of hawthorn, 3.0 kg of green tea.

[0060] Preparation method: 10.0 kg of Chinese wolfberry, 2.0 kg of ginseng, 5.0 kg of hawthorn, and 3.0 kg of green tea, a total of 20 kg of raw materials, are crushed into coarse powder, soaked in 20% ethanol for 1 hour, heated under reflux with 6 times the weight of 20% ethanol for extraction, extracted 3 times, 1 hour each time, concentrated to 1 g of crude drug per milliliter, the ethanol concentration is increased to 50%, filtered / centrifuged, and the filtrate is concentrated and dried under reduced pressure in vacuum to obtain 3.9 kg of the extract.

[0061] Example 4. Preparation of the traditional Chinese medicine composition powder (FHGL)

[0062] The steps are as follows:

[0063] (1) Preparation of wolfberry extract (LBE): 1.0 kg of Chinese wolfberry is soaked in water for 1 hour, extracted with 6 times the amount of water of the raw materials, concentrated and dried to obtain 213 g of wolfberry extract.

[0064] (2) Preparation of ginseng extract (PGE): 0.2 kg of ginseng is soaked in water for 0.5 hour, extracted with 6 times the amount of water of the raw materials, concentrated to 1.0 g of crude drug per milliliter, ethanol is added to the concentrated solution to a concentration of 50%, filtered / centrifuged, and the filtrate is concentrated and dried to obtain 38.5 g of ginseng extract.

[0065] (3) Preparation of hawthorn extract (HE): 0.4 kg of hawthorn is crushed and extracted with 8 times the amount of water of the raw materials, concentrated to 0.6 g of crude drug per milliliter, ethanol is added to the concentrated solution to a concentration of 30%, filtered / centrifuged, and the filtrate is concentrated and dried to obtain 105.8 g of hawthorn extract.

[0066] (4) Preparation of green tea extract (GTE): 0.6 kg of green tea is extracted with 10 times the amount of water of the raw materials, concentrated to 0.9 g of crude drug per milliliter, ethanol is added to the concentrated solution to a concentration of 50%, filtered / centrifuged, and the filtrate is concentrated and dried to obtain 98.2 g of green tea extract.

[0067] (5) Preparation of composition A (LBE + PGE): The wolfberry extract and ginseng extract are respectively crushed, passed through an 80-mesh sieve, and mixed evenly according to the mass ratio of wolfberry extract to ginseng extract = 5:1 to obtain it.

[0068] (6) Preparation of composition B (LBE + PGE + HE + GTE): The hawthorn extract and green tea extract are respectively crushed, passed through an 80-mesh sieve, and mixed with composition A so that the mass ratio of wolfberry extract, ginseng extract, hawthorn extract, and green tea extract is 5:1:2.5:2.5 to obtain it.

[0069] (7) Traditional Chinese medicine composition powder (FHGL): After mixing an appropriate amount of sucralose and inulin (a commercially available product) in equal increments, they are cross-fed into a two-dimensional mixing tank with the above-mentioned partial composition B; the mixed material is sieved (the mesh number of the sieve is 40 meshes), and the sieved material is fed into the mixer again for mixing for 40 minutes to obtain the traditional Chinese medicine composition powder FHGL; in this powder, the mass ratio of wolfberry extract, ginseng extract, hawthorn extract, green tea extract to inulin = 5:1:2.5:2.5:14.

[0070] Test example 1. For 60 Effect of Co-γ ray irradiation on the reproduction and survival rate of N2 Caenorhabditis elegans

[0071] Using 60 The N2 Caenorhabditis elegans model of radiation damage induced by Co-γ ray irradiation was used to observe the effect of the traditional Chinese medicine composition powder (FHGL) on the reproduction and survival rate of irradiated N2 Caenorhabditis elegans. The results showed that FHGL could dose-dependently increase 60 The number of eggs laid by Co-γ ray irradiated Caenorhabditis elegans and the median survival period of Caenorhabditis elegans.

[0072] Experimental method:

[0073] 1. Preparation of 8 test substances

[0074] According to the preparation method provided in Example 4 above, wolfberry extract (LBE), ginseng extract (PGE), hawthorn extract (HE), green tea extract (GTE), composition A (LBE + PGE), composition B (LBE + PGE + HE + GTE), and traditional Chinese medicine composition powder (FHGL) were prepared.

[0075] Inulin: A commercially available product.

[0076] 2. Positive control drug: Amifostine (WR2721), a commercially available product.

[0077] 3. Preparation of bacteria-drug nematode medium

[0078] 3.1 Inoculate uracil-deficient Escherichia coli (OP50) into the nematode growth medium (NGM) and sterilize it under ultraviolet light for 1 h to obtain a bacteria-containing medium;

[0079] 3.2 Preparation of bacteria-drug nematode medium

[0080] The above 8 test substances were formulated into solutions with the concentrations shown in Table 1, sonicated for 2 min, and filtered through a 0.22 μm microporous membrane; weigh amifostine (WR2721) and dissolve it in distilled water to a final concentration of 0.1 mM, and filter it through a 0.22 μm microporous membrane; according to the same dosing dose, add 1 mL of each filtrate to the bacteria-containing medium prepared in "3.1".

[0081] Table 1 Final Concentrations of Test Substances

[0082]

[0083] 4. Synchronization of Caenorhabditis elegans

[0084] 4.1 Select the NGM medium with more eggs in the nematodes, and use M9 buffer (0.1476M Na2HPO 4， 0.037M KH2PO4, 0.425M NaCl) to rinse the medium, and transfer the nematodes to a centrifuge tube.

[0085] 4.2 Let the centrifuge tube stand until the nematodes precipitate, remove the supernatant, aspirate M9 buffer to continue rinsing, and remove the supernatant after precipitation. Repeat this step until the liquid is clear.

[0086] 4.3 Add freshly prepared nematode lysate (0.023M NaClO, 0.04M NaOH) to the centrifuge tube and shake vigorously until the nematodes are lysed to obtain eggs.

[0087] 4.4 Place it in a centrifuge, centrifuge to remove the supernatant, retain the precipitate, rinse with M9 buffer and then repeat the centrifugation operation 3 times.

[0088] 4.5 Transfer the eggs to a 35mm NGM culture dish without food and culture in a biochemical incubator at 20°C for 16h. At this time, the nematodes are in the L1 stage (at 20°C, the wild-type nematode N2 can be divided into the embryonic stage, larval stage and adult stage. Among them, the larval stage can be further divided into L1 stage, L2 stage, L3 stage and L4 stage. Among them, the first 3-4 days after developing into an adult are the egg-laying period).

[0089] 4.6 Transfer the L1-stage nematodes to a 90mm NGM medium coated with food, and this is counted as day 0.

[0090] 5. 60 Co-γ Ray Irradiation Conditions

[0091] Take the synchronized starved L1-stage Caenorhabditis elegans and inoculate them onto the plates with bacteria-drug. After feeding for 2h, irradiate with a total dose of 20Gy at 9.26Gy / min.

[0092] 6. Reproduction of Caenorhabditis elegans

[0093] Place three L4 larvae on a new NGM medium with bacteria-drug and culture at 20°C. Transfer three nematodes to a new NGM medium with bacteria-drug every day until they stop laying eggs. Count the number of offspring on the old medium about 24 hours after each transfer. Finally, count the total number of eggs laid by each nematode in a group during its lifetime on average, and repeat the experiment more than three times to verify the reliability of the results.

[0094] 7. Lifespan of Caenorhabditis elegans

[0095] The hatching of synchronized nematode eggs is recorded as the 0th day of the beginning of the nematode's life. The death of nematodes is recorded starting from the 10th day for lifespan statistics until all nematodes die (30 nematodes per group, repeated 3 times). The criterion for nematode death is no response to stimuli. Lost nematodes and those that died due to climbing onto the culture dish wall should be excluded from the statistical data.

[0096] Table 2 Effects of each extract and FHGL on 60 Experimental results of the effect of Co-γ ray radiation damage on the egg-laying number of Caenorhabditis elegans

[0097]

[0098] Experimental conclusion: As shown in Table 2, Figure 1 FHGL can significantly increase the egg-laying number of Caenorhabditis elegans damaged by Co-γ ray radiation, and the activity of the traditional Chinese medicine composition powder FHGL is stronger than that of each single extract, the LBE+PGE composition, and the LBE+PGE+HE+GTE composition. 60

[0099] Table 3 Effects of different concentrations of FHGL on 60 Experimental results of the effect of Co-γ ray radiation damage on the survival rate of Caenorhabditis elegans

[0100]

[0101] Experimental conclusion: As shown in Table 3 and Figure 2 FHGL can significantly increase the median survival time of Caenorhabditis elegans damaged by Co-γ ray radiation, and it shows a dose-dependent relationship. The anti-radiation ability of 10 mg / mL FHGL is equivalent to that of the positive drug WR2721 at 0.1 mM. 60

[0102] Test Example 2. Effect of FHGL on the reproduction of N2 Caenorhabditis elegans irradiated with X-rays

[0103] An N2-type Caenorhabditis elegans model with radiation damage caused by X-ray irradiation was used to observe the effect of FHGL on the reproduction of irradiated N2 Caenorhabditis elegans. The experiment showed that FHGL can significantly increase the egg-laying number of Caenorhabditis elegans damaged by X-ray radiation.

[0104] Experimental method:

[0105] Test substance: FHGL is the same as in Test Example 1; Positive control drug: Amifostine (WR2721).

[0106] 1. Preparation of bacteria-drug nematode culture medium

[0107] ​​1.1 Inoculate uracil - defective Escherichia coli (OP50) into NGM medium and sterilize it under ultraviolet light for 1 h.

[0108] 1.2 After sterilizing different concentrations of FHGL and the positive control drug, add them to the ultraviolet - sterilized NGM medium to prepare media with different concentrations of the test substance - bacteria.

[0109] 2. Synchronization of Caenorhabditis elegans

[0110] 2.1 Select NGM medium with a large number of eggs in Caenorhabditis elegans, rinse the medium with M9 buffer, and transfer the nematodes to a centrifuge tube.

[0111] 2.2 Let the centrifuge tube stand until the nematodes precipitate, remove the supernatant, aspirate M9 buffer to continue rinsing, and remove the supernatant after precipitation. Repeat this step until the liquid is clear.

[0112] 2.3 Add freshly prepared nematode lysis solution to the centrifuge tube and shake vigorously until the nematodes are lysed to obtain eggs.

[0113] 2.4 Place it in a centrifuge, centrifuge to remove the supernatant, retain the precipitate, rinse with M9 buffer and then repeat the centrifugation operation 3 times.

[0114] 2.5 Transfer the eggs to a 35 - mm NGM culture dish without food and culture them in a biochemical incubator at 20 °C for 16 h. At this time, the nematodes are in the L1 stage.

[0115] 2.6 Transfer the L1 - stage nematodes to a 90 - mm NGM medium coated with food, and this is counted as day 0.

[0116] 3. X - ray irradiation conditions

[0117] Inoculate synchronized starved L1 - stage Caenorhabditis elegans onto the plate with bacteria - drug, and after feeding for 2 h, irradiate with a total dose of 20 Gy at a rate of 1.175 Gy / min.

[0118] 4. Reproduction of Caenorhabditis elegans

[0119] Place one L4 larva on a new NGM medium with bacteria - drug and culture it at 20 °C. Transfer this nematode to a new NGM medium with bacteria - drug every day until it stops laying eggs. Count the number of offspring on the old medium about 12 hours after each transfer. Finally, sum up the total number of eggs laid by this worm in its lifetime. Use 3 nematodes in each group and repeat three times.

[0120] Table 4 Experimental results of the effect of FHGL on the number of eggs laid by X - ray - irradiated damaged Caenorhabditis elegans

[0121]

[0122] Experimental results: As shown in Table 4, Figure 3 FHGL can significantly increase the number of eggs laid by Caenorhabditis elegans with X-ray radiation damage, suggesting that FHGL has a preventive and therapeutic effect on X-ray radiation damage in Caenorhabditis elegans, and shows a dose-dependent relationship. The anti-radiation ability of 10 mg / mL FHGL is equivalent to that of 0.1 mM positive drug WR2721.

[0123] Test Example 3. Effect on 60 Survival rate of Coγ-ray irradiated mice

[0124] Using a lethal dose of 60 Coγ-ray irradiated C57BL / 6 mouse model to observe the effect of FHGL on the survival rate of irradiated mice. The experimental results showed that FHGL could improve the survival of model mice 30 days after irradiation.

[0125] Experimental method:

[0126] 1. Test substance: FHGL is the same as in Test Example 1.

[0127] 2. Animal grouping and whole body radiation

[0128] C57BL / 6 mice were randomly divided into 5 groups, including: 1 normal control group, 1 whole body radiation model control group, and three FHGL groups with low, medium, and high doses, 12 mice / group. The low, medium, and high doses of FHGL were 150 mg / kg, 300 mg / kg, and 600 mg / kg, respectively.

[0129] FHGL was intragastrically administered daily starting 5 days before radiation, and the intragastric volume each time was about 0.4 mL (the intragastric volume of each test substance was adjusted according to the actual body weight of the animals). The normal control group (without adding FHGL) and the radiation control group were intragastrically administered 0.4 mL of sterilized water for daily drinking of animals in parallel. Except for the normal control group, the radiation control group and each drug administration group received 60 Single whole body radiation with a Co-γ radiation source, the absorbed dose was 8.5 Gy, the dose rate was 67.51 cGy / min, the animals were 4.0 meters away from the radiation source, and intragastric administration continued for 21 days after radiation. The behavioral status of the test mice was observed daily, and the death situation within 30 days after radiation was recorded.

[0130] 3. Experimental results

[0131] 3.1 Physical signs and behavioral changes

[0132] Compared with the normal control group, the body temperature of the mice in the radiation group increased slightly on the day of irradiation, and they were hyperactive and manic. The mice in the radiation model group gradually lost their vitality starting from the second day, showing slow movement, listlessness, and their hair color gradually became dark gray. After about one week, the back hair was dull, sparse, and messy; the back hair glossiness and behavioral status of the mice in the FHGL group were better than those in the radiation model group.

[0133] 3.2 Survival rate

[0134] As shown in Table 5 below, after irradiation with 8.5 Gy γ-rays, all C57BL / 6 mice in the whole-body radiation model control group died within 19 days after radiation, and mice in each test group of FHGL showed better survival rates.

[0135] During the 30-day observation period after radiation, FHGL could improve the survival rate of irradiated mice. Among the low (150 mg / kg), medium (300 mg / kg), and high-dose (600 mg / kg) groups of FHGL, 2, 4, and 5 mice survived respectively. The experiment showed that FHGL could reduce the radiation damage of 8.5 Gy γ-rays to C57BL / 6 mice.

[0136] Table 5 Experimental results of the effect of FHGL on the survival rate of mice with γ-ray radiation damage

[0137]

[0138] Test Example 4. Effect on the survival rate of X-ray irradiated mice

[0139] A BALB / C mouse model with lethal-dose X-ray radiation damage was used to observe the effect of the traditional Chinese medicine composition on the survival rate of irradiated mice. The experimental results showed that the traditional Chinese medicine composition could improve the survival of model mice 30 days after irradiation.

[0140] Experimental method:

[0141] 1. Preparation of the test substance

[0142] The preparation steps of the traditional Chinese medicine composition are as follows: 3 kg of wolfberry fruits, 0.5 kg of ginseng, 1.5 kg of hawthorn fruits, and 1.5 kg of green tea, a total of 6.5 kg of raw materials, were crushed into coarse powder, soaked in 20% ethanol for 1 hour, refluxed and extracted 3 times with 6 times the amount of 20% ethanol for 2 hours each time, concentrated to 1 g of crude drug / ml, the ethanol concentration was increased to 60%, filtered / centrifuged, and the filtrate was concentrated and dried under reduced pressure in a vacuum to obtain 1.46 kg of the traditional Chinese medicine composition.

[0143] 2. Animal grouping and whole-body radiation

[0144] BALB / C mice were randomly divided into 5 groups, including: 1 normal control group, 1 whole-body radiation model control group, and low, medium, and high doses of the traditional Chinese medicine composition, with 10 mice in each group.

[0145] The low, medium, and high doses of the traditional Chinese medicine composition were 200 mg / kg, 400 mg / kg, and 800 mg / kg respectively. The test substances were intragastrically administered daily starting 7 days before radiation, with each intragastric administration volume being approximately 0.4 mL (the intragastric administration volume of each test substance was adjusted according to the actual body weight of the animals). The normal control group and the radiation control group were intragastrically administered 0.4 mL of sterilized water for daily drinking of the animals in parallel every day.

[0146] Except for the control groups, the animals in the other groups were subjected to a single whole-body X-ray irradiation (administered after irradiation on the same day), and the test substances or solvents were continuously administered for 21 days after radiation. The irradiation dose was 6.0 Gy, and the irradiation dose rate was 190.7 cGy / min. The behavioral status of the test mice was observed daily, and the death situation within 30 days after radiation was recorded.

[0147] 3. Experimental results

[0148] 3.1 Physical signs and behavioral changes

[0149] Compared with the normal control group, the body temperature of the mice in the radiation group slightly increased on the day of irradiation, and they were hyperactive and manic. The mice in the radiation model group gradually lost their vitality starting from the next day, showing slow movement and listlessness. After about 7 days, the dorsal hair was sparse and had poor gloss; the dorsal hair gloss and behavioral status of the mice in the traditional Chinese medicine composition group were better than those in the radiation model group.

[0150] 3.2 Survival rate

[0151] As shown in the following table, after 6.0 Gy X-ray irradiation, all the BALB / C mice in the whole-body radiation model control group died within 13 days after radiation. The BALB / C mice in each test substance group of the traditional Chinese medicine composition showed better survival rates. During the 30-day observation period after radiation, the traditional Chinese medicine composition could improve the survival rate of the irradiated mice. Among them, 2, 4, and 4 mice survived in the low (200 mg / kg), medium (400 mg / kg), and high-dose (800 mg / kg) groups of the traditional Chinese medicine composition respectively. The above experimental results indicate that the traditional Chinese medicine composition can reduce the radiation damage of 6.0 Gy X-ray to BALB / C mice.

[0152] Table 6 Experimental results of the effect of traditional Chinese medicine composition on the survival rate of mice with X-ray radiation injury

[0153]

[0154] Test example 5. Effect on 60 The body weight and peripheral blood picture of mice irradiated with

[0155] Using a sub-lethal dose of 60C57BL / 6 mouse model with Coγ-ray radiation injury was used to observe the effects of FHGL on the body weight and peripheral blood picture of irradiated mice. The experimental results showed that FHGL could promote the recovery of body weight and peripheral blood picture in the model mice after radiation.

[0156] Experimental method:

[0157] 1. Test substance: FHGL as in Test Example 1.

[0158] 2. Animal grouping and whole-body radiation

[0159] C57BL / 6 mice were randomly divided into 3 groups, including: 1 normal control group, 1 whole-body radiation model control group, and 1 FHGL group at a dose of 300 mg / kg, with 10 mice in each group. FHGL was administered by gavage daily starting from 7 days before radiation, and the volume of each gavage was approximately 0.4 mL (the gavage volume of each test substance was adjusted according to the actual body weight of the animals). The normal control group (without adding FHGL) and the radiation control group were gavaged with 0.4 mL of sterilized water for daily drinking of animals in parallel. Except for the normal control group, the mice in other groups received 60 a single whole-body irradiation with Coγ-rays at a dose of 6.0 Gy, an irradiation dose rate of 61.2 cGy / min. After radiation, gavage continued for 21 days, once a day. The body weight was recorded daily during the experiment. Blood was collected from the retro-orbital venous plexus at the blank point, 1 d, 3 d, 7 d, 14 d, and 21 d after irradiation, placed in an EDTA anticoagulant EP tube, shaken well, 20 μL of anticoagulated whole blood was aspirated and added to the diluent, and after mixing, it was detected with an automatic blood cell analyzer.

[0160] 3. Experimental results

[0161] 3.1 Effects on mouse body weight:

[0162] As Figure 4 shown, after 6.0 Gy γ-ray irradiation, the body weight of C57BL / 6 mice decreased significantly and reached the lowest on the 2nd - 3rd day. The body weights of mice in the whole-body radiation group (model group) and the FHGL group were significantly lower than those in the normal control group. From 3 d to 10 d after irradiation, the body weights of irradiated mice showed a slow upward trend, and the body weights of mice in the FHGL group recovered faster. The body weight of irradiated mice decreased again on the 11th day after radiation and then gradually increased. From 5 d to 14 d after irradiation, the FHGL group showed a trend of promoting the recovery of mouse body weight.

[0163] 3.2 Effects on peripheral blood cells

[0164] 3.2.1 Percentage of lymphocytes

[0165] A decrease in the percentage of lymphocytes (LY%) is commonly seen in immune damage, the acute phase of certain infectious diseases, and after exposure to certain chemicals and radiation. Early recovery of lymphocyte loss is very important for the immune function of the host after radiation.

[0166] As Figure 5 shown, after 6.0 Gy γ-ray irradiation, the percentage of peripheral blood lymphocytes (LY%) in the irradiated model group of mice decreased rapidly on day 1 after irradiation, and the difference was statistically significant compared with the normal control group (p < 0.001). After that, LY% began to rise. The recovery of LY% in the model group of mice was slower and remained at a lower level within 21 days after irradiation. FHGL intervention significantly alleviated the decrease in LY% after irradiation (p < 0.001) and significantly accelerated the recovery of LY%. On day 10 after irradiation, the LY% of the mice in the FHGL group had recovered to the normal level, and there was no significant difference compared with the control group. From day 10 to day 21 after irradiation, the peripheral blood LY% in the FHGL group remained stable at the normal level, with no significant difference compared with the non-irradiated normal group (p > 0.05), and a significant difference compared with the model group (p < 0.05), indicating that FHGL can significantly reduce the decrease in peripheral blood LY% in C57BL / 6 mice after radiation and play a promoting role in the recovery of the immune function of the body after radiation.

[0167] 3.2.2 Percentage of neutrophils

[0168] As Figure 6 shown, after 6.0 Gy γ-ray irradiation of C57BL / 6 mice, the percentage of peripheral blood neutrophils (NE%) increased rapidly on day 1 after irradiation, and the difference was statistically significant compared with the normal control group (p < 0.001). After that, NE% began to decline. The recovery of NE% in the model group of mice was slower. FHGL treatment significantly alleviated the increase in NE% after irradiation (p < 0.001) and promoted the rapid recovery of NE%. On day 3 after irradiation, the peripheral blood NE% of the mice in the model group was still significantly higher than that of the normal group (p < 0.001), while the NE% of the mice in the FHGL group had basically recovered to the normal level. The NE% in the model group increased again on day 21 after irradiation, and the difference was statistically significant compared with the normal control group (p < 0.05). The peripheral blood NE% in the FHGL group remained at the normal level, and the difference was statistically significant compared with the model group (p < 0.001), indicating that FHGL can significantly relieve the change in peripheral blood NE% in C57BL / 6 mice caused by radiation.

[0169] 3.2.3 Percentage of monocytes

[0170] As Figure 7As shown, after C57BL / 6 mice were irradiated with 6.0 Gy of γ-rays, the percentage of peripheral blood monocytes (MO%) slowly increased after irradiation and was significantly different from that of the normal control group at 7 days after irradiation (p < 0.001). The MO% in the peripheral blood of the mice in the model group increased significantly from 10 days to 14 days after irradiation and gradually recovered from 14 days to 21 days after irradiation. FHGL treatment alleviated the increase in MO% after irradiation, and the MO% level was significantly lower than that of the model group at 10 days and 14 days after irradiation (p < 0.05), indicating that FHGL can significantly reduce the change in MO% in the peripheral blood of model mice caused by irradiation.

[0171] Experimental conclusion: In this invention, the Caenorhabditis elegans model of radiation injury irradiated with γ-rays, the Caenorhabditis elegans model of radiation injury irradiated with X-rays, the mouse model of radiation injury irradiated with γ-rays, and the mouse model of radiation injury irradiated with X-rays were respectively used to observe the effects of the traditional Chinese medicine composition on the number of eggs laid by Caenorhabditis elegans with radiation injury, the median survival time, the survival rate of irradiated mice, the body weight of irradiated mice, and the peripheral blood picture. The results showed that the traditional Chinese medicine composition could significantly increase the number of eggs laid and the median survival time of Caenorhabditis elegans with radiation injury irradiated with γ-rays, X-rays, etc., could significantly increase the survival rate of irradiated mice, promote the recovery of the body weight of model mice after irradiation, and promote the recovery of the peripheral blood picture. The above research results provide a theoretical basis for the further development of new natural radiation protectants.

[0172] Although the present invention has been described in detail with general descriptions and specific embodiments above, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. A traditional Chinese medicine composition is prepared from raw materials in the following parts by weight: 3 - 6 parts by weight of wolfberry fruits, 1 part by weight of ginseng, 1 - 3 parts by weight of hawthorn, and 1 - 3 parts by weight of green tea.

2. The traditional Chinese medicine composition according to claim 1, wherein: The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 10 parts by weight of wolfberry fruits, 3 parts by weight of ginseng, 3 parts by weight of hawthorn fruits, and 5 parts by weight of green tea; Alternatively, the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 8 parts by weight of wolfberry fruits, 2 parts by weight of ginseng, 4 parts by weight of hawthorn fruits, and 4 parts by weight of green tea; Alternatively, the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 10 parts by weight of wolfberry fruits, 2 parts by weight of ginseng, 5 parts by weight of hawthorn fruits, and 3 parts by weight of green tea.

3. The preparation method of the traditional Chinese medicine composition according to claim 1 or 2 comprises the following steps: subjecting each raw material to alcohol extraction or water extraction to obtain the composition.

4. The preparation method of the traditional Chinese medicine composition according to claim 3, wherein: The reagent used for alcohol extraction is an aqueous ethanol solution with a volume concentration not exceeding 50%; The method of alcohol extraction is reflux extraction or percolation extraction.

5. The preparation method of the traditional Chinese medicine composition according to claim 3, wherein: The method of water extraction is decoction extraction or reflux extraction.

6. A traditional Chinese medicine composition is composed of the following components in parts by weight: 4.5 - 6.5 parts by weight of wolfberry fruit extract, 1 part by weight of ginseng extract, 2 - 3.5 parts by weight of hawthorn fruit extract, 2 - 3 parts by weight of green tea extract, and 12.5 - 15 parts by weight of inulin.

7. A traditional Chinese medicine preparation comprises the traditional Chinese medicine composition according to claim 1 or 2 or the traditional Chinese medicine composition according to claim 6.

8. The traditional Chinese medicine preparation according to claim 7, wherein: The dosage form of the traditional Chinese medicine preparation is powder, granule, oral liquid, capsule, tablet, or effervescent tablet.

9. The application of the traditional Chinese medicine composition according to claim 1 or 2, the traditional Chinese medicine composition according to claim 6, and the traditional Chinese medicine preparation according to claim 7 or 8 in the prevention or treatment of preparing radiation damage protection products.

10. The application according to claim 9, wherein: The product includes food, health care product, or pharmaceutical preparation; The radiation damage includes one or more of the damages caused by α - rays, β - rays, γ - rays, X - rays, ultraviolet rays, microwaves, and radio waves.

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