Preparation method of Trillium japonicum extract and its application in resisting radiation damage

Abstract

The present invention provides a method for preparing a Trillium extract, comprising: step 1: crushing the rhizome of Trillium to obtain Trillium powder; step 2: soaking the Trillium powder in a first ethanol solution, decocting or refluxing extraction, filtering, collecting the filtrate, concentrating and drying to obtain the Trillium extract, wherein the concentration of the first ethanol solution is 0 to 75% by volume. The Trillium extract obtained by the preparation method of the present invention has the effect of preventing and treating radiation damage and has good application prospects.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a preparation method of a Trillium extract and an application thereof in resisting radiation damage. Background Art

[0002] Trillium is a precious Chinese medicine from the genus Trillium of the Liliaceae family, and is also one of the famous and commonly used medicinal flavors of the Tujia ethnic group. The plants of this genus mainly include Trillium tschonoskii Maxim., Trilliumkamtschaticum Pall.ex Purs, and Trillium govanianum Wall.ex Royle. Existing studies have shown that Trillium has good anti-tumor, anti-inflammatory, immunomodulatory, and anti-aging effects on improving rat learning and memory functions. It is mainly used clinically to treat dizziness, insomnia, traumatic injuries, traumatic bleeding, neurasthenia, hypertension, sequelae of concussion, and other diseases.

[0003] With the development of science and technology and the widespread application of nuclear technology, ionizing radiation has penetrated into all areas of life. For example, the application of nuclear energy technology is becoming more and more widespread; in clinical practice, during medical diagnosis and treatment, especially during tumor radiotherapy, the healthy tissues of patients will also be damaged by radiation to a certain extent. While ionizing radiation brings convenience to people, it also has a certain impact on health. It can produce reactive oxygen (ROS) free radicals through tissues and cells, interfere with macromolecules such as DNA and proteins, induce cell damage and abnormal cell function, and cause damage to the human and animal body, such as the hematopoietic system, immune system and digestive system, and ultimately lead to functional disorders of multiple organs of the body, lesions and even death. With people's in-depth understanding of radiation damage and protection, many radiation protectors have been discovered. Radiation protectors are divided into two categories: one is mainly artificially synthesized drugs, mainly tryptamine compounds, sulfur-containing compounds, etc., but this type of radiation protector has disadvantages such as large side effects, short duration of action, and high price; the other is natural radiation protectors, including plant polyphenols, alkaloids, saponins, anthocyanins, vitamins, etc., which have the advantages of long duration of action, low cost and no obvious toxic side effects.

[0004] Different tissues and cells in the human body have different sensitivities to radiation. Among them, the intestine is one of the tissues that is sensitive to radiation damage. High-dose ionizing radiation can easily induce acute gastrointestinal syndrome (AGS), which greatly threatens the survival of the irradiated population. Clinically, there is no effective drug with few adverse reactions to prevent or treat AGS induced by high-dose ionizing radiation. Finding new natural radiation protectors that are highly effective, non-toxic, and can be taken for a long time to prevent and / or treat radiation damage to intestinal tissue has become the current research focus. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, one object of the present invention is to provide a preparation method of a Trillium extract, a Trillium extract and an application thereof, wherein the Trillium extract obtained by the preparation method has the effect of preventing and treating radiation damage and has good application prospects.

[0006] In one aspect of the present invention, the present invention provides a method for preparing a Trillium extract. According to an embodiment of the present invention, the method comprises: step 1: crushing the rhizome of Trillium to obtain Trillium powder; step 2: soaking the Trillium powder with a first ethanol solution, decocting or refluxing extraction, filtering, collecting the filtrate, concentrating and drying to obtain the Trillium extract, wherein the concentration of the first ethanol solution is 0 to 75% by volume. The Trillium extract obtained by the method according to the embodiment of the present invention can extract the effective ingredients in Trillium, such as oligosaccharides, and the obtained extract has the effect of preventing and treating radiation damage.

[0007] According to an embodiment of the present invention, the preparation method of the above-mentioned Trillium extract may also have the following additional technical features:

[0008] According to an embodiment of the present invention, before the concentration and drying, the filtrate is further subjected to the following operation: the filtrate is added to a chromatography column, eluted with a second ethanol solution, the effluent is collected, and the concentrated solution is concentrated and dried to obtain the Trillium extract, wherein the concentration of the second ethanol solution is 0 to 30% by volume, and more preferably, the concentration of the second ethanol solution is 0 to 20% by volume.

[0009] The inventors found that column chromatography can be used to better separate the extract with radiation damage protection. Furthermore, the inventors found that the oligosaccharides in the filtrate have an excellent effect on the protection of radiation damage. Then, in order to obtain purer oligosaccharides, the inventors further studied and found that after elution with 0-30% by volume ethanol solution, the oligosaccharide content in the effluent was high, and thus the obtained extract had a good protection effect on radiation damage.

[0010] According to an embodiment of the present invention, before the concentration and drying, the effluent is added to the chromatography column again, eluted with a third ethanol solution, the effluent is collected, and the concentrated solution is concentrated and dried to obtain the Trillium extract, wherein the concentration of the third ethanol solution is 0 to 20% by volume. Thus, the obtained oligosaccharides are purified again to further improve the purity of the oligosaccharides in the extract.

[0011] According to an embodiment of the present invention, the chromatography column is filled with a macroporous adsorption resin, preferably, the macroporous adsorption resin is a non-polar or weakly polar macroporous adsorption resin, more preferably AB-8, D-101, SP825 or HP20. Thus, other components in the filtrate except oligosaccharides can be better adsorbed, so that an oligosaccharide-rich extract can be better obtained.

[0012] According to an embodiment of the present invention, the medicinal material source of the Trillium rhizome is a plant of the genus Trillium in the Liliaceae family, including but not limited to one or more of Trillium tschonoskii Maxim., Trillium kamtschaticum Pall.ex Pursh and Trillium govanianum Wall.ex Royle.

[0013] In another aspect of the present invention, the present invention provides a Trillium extract. According to an embodiment of the present invention, the Trillium extract is obtained by the preparation method of the Trillium extract described above. The Trillium extract is rich in components with radiation damage protection effects, such as oligosaccharides, has the effects of preventing and treating radiation damage, and has good application prospects.

[0014] In another aspect of the present invention, the present invention proposes the use of the above-mentioned Trillium extract in the preparation of an anti-radiation damage product. The Trillium extract can be used for the prevention and treatment of radiation damage, and has a good application prospect.

[0015] According to an embodiment of the present invention, the radiation damage is ionizing radiation damage; the ionizing radiation damage includes but is not limited to one or more of α rays, β rays, γ rays and X rays.

[0016] According to an embodiment of the present invention, the ionizing radiation damage is intestinal damage caused by ionizing radiation.

[0017] According to an embodiment of the present invention, the anti-radiation damage product includes but is not limited to food, health food or medicine.

[0018] According to an embodiment of the present invention, the dosage form of the drug is granules, oral liquid, capsule, tablet, effervescent tablet, powder injection, water injection or injection.

[0019] According to an embodiment of the present invention, the medicine includes a single dosage form or a compound dosage form.

[0020] Beneficial effects:

[0021] The present invention extracts active ingredients (mainly oligosaccharides) with radiation damage protection from the natural medicine Trillium, which has good biocompatibility and has obvious protective effects on mouse injury models irradiated by gamma rays and X-rays, especially on small intestine damage irradiated by gamma rays and has strong biological activity. The extract of the present invention can be made into radiation-resistant products, such as food, medicine or health products, and has good application prospects.

[0022] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0024] Figure 1 To investigate the effects of purified oligosaccharides (TT-2-0) and (TT-2-15) from Trillium on the clonogenicity of γ-ray irradiated IEC cells.

[0025] Figure 2 The oligosaccharide part of Trillium (TT-2) inhibits the apoptosis of IEC cells irradiated by gamma rays;

[0026] Figure 3 The oligosaccharide part of Trillium (TT-2) promotes the proliferation of IEC cells irradiated by gamma rays. DETAILED DESCRIPTION

[0027] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0028] Example 1

[0029] A method for preparing a Trillium extract: crush 20 kg of Trillium rhizomes into coarse powder, soak in 50% ethanol for 1 hour, heat and reflux extract with 10 times the amount of 50% ethanol for 3 times, 2 hours each time, filter, and combine the filtrate. Concentrate the filtrate and dry it to obtain 5.7 kg of a crude Trillium extract;

[0030] Example 2

[0031] A method for preparing a Trillium extract: 5 kg of Trillium rhizomes are crushed into coarse powder, soaked in 70% ethanol for 1 hour, heated and refluxed for extraction for 3 times with 8 times the amount of 70% ethanol, each time for 2 hours, filtered, and the filtrate is combined. The filtrate is concentrated and vacuum dried to obtain 1.1 kg of a crude Trillium extract;

[0032] Example 3

[0033] A method for preparing a Trillium extract: 5 kg of Trillium rhizomes are crushed into coarse powder, soaked in water for 1 hour, heated and extracted with 8 times the amount of water for 3 times, each time for 2 hours, filtered, and the filtrate is combined. The filtrate is concentrated and freeze-dried to obtain 2.2 kg of a crude Trillium extract;

[0034] Example 4

[0035] A method for preparing oligosaccharide fraction of Trillium: crush 6 kg of Trillium rhizome into coarse powder, soak in 50% ethanol for 1 hour, heat and reflux extract with 10 times the amount of 50% ethanol for 3 times, 2 hours each time, filter, and combine the filtrate. The above filtrate is concentrated under reduced pressure at 60-70°C to 1.0 g of raw medicinal material / ml, refrigerated at 0-4°C and left to stand overnight, filtered, and the filtrate is enriched on an AB-8 macroporous adsorption resin column, eluted with 15% ethanol to obtain an eluate, concentrated under reduced pressure, and the concentrate is dried with a freeze dryer. The freeze-dried powder is collected, which is the oligosaccharide fraction of Trillium, a total of 280.2 g;

[0036] Example 5

[0037] A method for preparing oligosaccharide fraction of Trillium: crush 3kg of Trillium rhizome into coarse powder, soak in 75% ethanol for 1 hour, heat and reflux extract with 12 times the amount of 75% ethanol for 3 times, 2 hours each time, filter, and combine the filtrate. The above filtrate is concentrated under reduced pressure at 60-70°C to 1.0g of raw medicinal material / ml, refrigerated at 0-4°C overnight, filtered, and the filtrate is enriched on a D101 macroporous adsorption resin column, eluted with 30% ethanol to obtain an eluate, and the eluate is concentrated under reduced pressure, and the concentrated solution is vacuum dried under reduced pressure, and the dry powder collected is the oligosaccharide fraction of Trillium, a total of 160.4g;

[0038] Example 6

[0039] A method for preparing oligosaccharide fraction of Trillium: crush 5kg of Trillium rhizome into coarse powder, soak in water for 1 hour, heat and extract with 8 times the amount of water for 3 times, 2 hours each time, filter, and combine the filtrate. The filtrate is concentrated under reduced pressure at 60-70°C to 1.0g of raw medicinal material / ml, refrigerated at 0-4°C overnight, filtered, and the filtrate is enriched on a SP825 macroporous adsorption resin column, eluted with 20% ethanol to obtain an eluate, and the eluate is concentrated under reduced pressure. The concentrate is dried with a freeze dryer, and the freeze-dried powder is collected, which is the oligosaccharide fraction of Trillium, a total of 340.5g;

[0040] Example 7

[0041] A method for preparing purified oligosaccharides of Trillium: 5 kg of Trillium rhizomes are crushed into coarse powder, soaked in 50% ethanol for 1 hour, heated and refluxed with 10 times the amount of 50% ethanol for extraction 3 times, each time for 2 hours, filtered, and the filtrate is combined. The filtrate is concentrated under reduced pressure at 60-70°C to 1.0 g of raw medicinal material / ml, refrigerated at 0-4°C overnight, filtered, the filtrate is enriched on a SP825 macroporous adsorption resin column, eluted with 15% ethanol, and an eluate is obtained. The eluate is concentrated under reduced pressure, the concentrated solution is again subjected to HP20 macroporous adsorption resin, eluted with water, the water elution part is concentrated and then dried with a freeze dryer, and the freeze-dried powder is collected, which is the purified oligosaccharides of Trillium, a total of 30.2 g.

[0042] Example 8

[0043] A method for preparing purified oligosaccharides of Trillium: 5 kg of Trillium rhizomes are crushed into coarse powder, soaked in 50% ethanol for 1 hour, heated and refluxed with 10 times the amount of 50% ethanol for extraction 3 times, each time for 2 hours, filtered, and the filtrate is combined. The filtrate is concentrated under reduced pressure at 60-70°C to 1.0 g of raw medicinal material / ml, refrigerated at 0-4°C overnight, filtered, the filtrate is enriched on a SP825 macroporous adsorption resin column, eluted with 15% ethanol, and an eluate is obtained. The eluate is concentrated under reduced pressure to a small volume (about 800 ml), the concentrated solution is again subjected to SP825 macroporous adsorption resin, eluted with 10% ethanol, and then partially concentrated and dried with a freeze dryer, and the freeze-dried powder is collected, which is the purified oligosaccharide of Trillium, a total of 40.6 g.

[0044] Example 9

[0045] A C57BL / 6 mouse model was damaged by a lethal dose of γ-ray radiation to observe the effects of Trillium extract and Jilin Trillium extract on the survival rate of irradiated mice. The experimental results showed that Trillium extract and Jilin Trillium extract could improve the survival of model mice 30 days after irradiation.

[0046] 1. Preparation of test substances

[0047] a. Preparation of Trillium extract: Grind 2 kg of Trillium rhizome into coarse powder, soak in 50% ethanol for 1 hour, heat and reflux with 10 times the amount of solvent for 3 times, 2 hours each time, filter, and combine the filtrate. Concentrate the filtrate, and dry under reduced pressure to obtain Trillium extract. Prepare the above Trillium extract into an aqueous solution / suspension of appropriate concentration for use;

[0048] b. Preparation of Jilin Trillium extract: 3 kg of Jilin Trillium rhizome was crushed into coarse powder, soaked in 50% solvent for 1 hour, heated and extracted 3 times with 8 times the amount of solvent, each time for 2 hours, filtered, and the filtrate was combined. The filtrate was concentrated and vacuum dried under reduced pressure to obtain Jilin Trillium extract. The above Jilin Trillium extract was prepared into an aqueous solution / suspension of appropriate concentration for use.

[0049] 2. Animal Grouping and Whole-body Irradiation

[0050] C57BL / 6 mice were randomly divided into 8 groups, including: 1 normal control group, 1 whole-body radiation model control group, and low, medium and high doses of Trillium extract groups and low, medium and high doses of Jilin Trillium extract groups, 10 mice in each group. The low, medium and high doses of the Trillium extract group were 200 mg / kg, 400 mg / kg and 800 mg / kg, respectively; the low, medium and high doses of the Jilin Trillium extract group were 200 mg / kg, 400 mg / kg and 800 mg / kg, respectively. Starting from 7 days before radiation, the Trillium extract and Jilin Trillium extract test substances were gavaged daily, and the gavage volume each time was about 0.4 mL (the gavage volume of each test substance was adjusted according to the actual body weight of the animal). The normal control group (without the addition of Trillium extract) and the radiation control group were gavaged with 0.4 mL of the animal's daily drinking sterile water in parallel every day. Except for the normal control group, the radiation control group and each drug-treated group received 60 The whole body was irradiated with Co-γ radiation source at one time, with an absorbed dose of 8.5 Gy and a dose rate of 67.51 cGy / min. The animals were 4.0 meters away from the radiation source. The mice were gavaged continuously for 21 days after irradiation. The behavioral status of the mice was observed daily, and deaths within 30 days after irradiation were recorded.

[0051] 3. Experimental results

[0052] 3.1 Physical signs and behavioral changes

[0053] Compared with the normal control group, the mice in the radiation group had a slightly higher body temperature and manic behavior on the day of irradiation. The mice in the radiation model group began to gradually lose vitality on the second day, showing slow movements and lack of energy. Their fur color gradually darkened. After one week, the back fur was sparse, messy, and had poor glossiness. The mice in the Trillium extract group and the Jilin Trillium extract group had better back fur glossiness and behavior than the radiation model group.

[0054] 3.2 Survival rate

[0055] As shown in Table 1 below, after 8.5Gy γ-ray irradiation, all C57BL / 6 mice in the whole body radiation model control group died within 19 days after irradiation, and mice in the Trillium extract group and the Jilin Trillium extract test group showed a good survival rate. During the 30-day observation period after irradiation, the Trillium extract group and the Jilin Trillium extract group can improve the survival rate of irradiated mice, among which the low (200mg / kg), medium (400mg / kg), and high dose (800mg / kg) groups of the Trillium extract group had 2, 4, and 5 mice surviving, respectively, and the low (200mg / kg), medium (400mg / kg), and high dose (800mg / kg) groups of the Jilin Trillium extract group had 2, 4, and 3 mice surviving, respectively. The above experimental results show that the Trillium extract group and the Jilin Trillium extract can reduce the radiation damage of 8.5Gy γ-rays to C57BL / 6 mice.

[0056] Table 1 Survival rate experimental results of Trillium and Jilin Trillium extract

[0057]

[0058]

[0059] Example 10

[0060] A BALB / C mouse model was damaged by a lethal dose of X-ray radiation to observe the effects of Trillium extract and Jilin Trillium extract on the survival rate of irradiated mice. The experimental results showed that Trillium extract and Jilin Trillium extract could improve the survival of model mice 30 days after irradiation.

[0061] 1. Preparation of test substances

[0062] a. Preparation of Trillium extract: Grind 5 kg of Trillium rhizome into coarse powder, soak in water for 1 hour, heat and extract with 12 times the amount of solvent for 3 times, 2 hours each time, filter, and combine the filtrate. Concentrate the filtrate, and dry under reduced pressure to obtain Trillium extract. Prepare the above Trillium extract into an aqueous solution of appropriate concentration for later use;

[0063] b. Preparation of Jilin Trillium extract: 3 kg of Jilin Trillium rhizome was crushed into coarse powder, soaked in water for 1 hour, heated and extracted 3 times with 10 times the amount of solvent, each time for 2 hours, filtered, and the filtrate was combined. The filtrate was concentrated and vacuum dried under reduced pressure to obtain Jilin Trillium extract. The above Jilin Trillium extract was prepared into an aqueous solution of appropriate concentration for standby use.

[0064] 2. Animal Grouping and Whole-body Irradiation

[0065] BALB / C mice were randomly divided into 8 groups, including 1 normal control group, 1 whole body radiation model control group, and 3 doses of Trillium extract group, 10 mice / group. The 3 doses of Trillium extract group were 150mg / kg, 300mg / kg, 600mg / kg respectively; the 3 doses of Jilin Trillium extract were 150mg / kg, 300mg / kg, 600mg / kg respectively. Trillium extract and Jilin Trillium extract were gavaged daily from 7 days before radiation, and the gavage volume was about 0.4mL each time (the gavage volume of each test substance was adjusted according to the actual body weight of the animal). The normal control group and the radiation control group were gavaged with 0.4mL of sterile drinking water for animals every day.

[0066] Except for the control group, the animals in the other groups were subjected to one-time X-ray whole-body irradiation (given after irradiation on the same day), and the test substance or solvent was continued to be given for 21 days after irradiation. The irradiation dose was 6.0Gy, and the irradiation dose rate was 190.7cGy / min. The behavior of the test mice was observed daily, and the death within 30 days after irradiation was recorded.

[0067] 3. Experimental results

[0068] 3.1 Physical signs and behavioral changes

[0069] Compared with the normal control group, the mice in the radiation group had a slightly higher body temperature and manic behavior on the day of irradiation. The mice in the radiation model group began to lose vitality on the second day, showing slow movements and lack of energy. After about 7 days, the back hair was sparse and the glossiness was poor. The mice in the Trillium extract group and the Jilin Trillium extract group had better back hair gloss and behavior than the radiation model group.

[0070] 3.2 Survival rate

[0071] As shown in Table 2 below, after 6.0Gy X-ray irradiation, all BALB / C mice in the whole body radiation model control group died within 12 days after irradiation, and the BALB / C mice in the Trillium extract group and the Jilin Trillium extract test group showed a good survival rate. During the 30-day observation period after irradiation, the Trillium extract group and the Jilin Trillium extract group could improve the survival rate of irradiated mice, among which 3, 4 and 5 mice survived in the low (150mg / kg), medium (300mg / kg) and high (600mg / kg) dose groups of the Trillium extract group, respectively, and 2, 4 and 4 mice survived in the low (150mg / kg), medium (300mg / kg) and high (600mg / kg) dose groups of the Jilin Trillium extract group, respectively. The above experimental results show that the Trillium extract group and the Jilin Trillium extract can reduce the radiation damage of 6.0Gy X-rays to BALB / C mice.

[0072] Table 2 Survival rate experimental results of Trillium and Jilin Trillium extract

[0073]

[0074]

[0075] Embodiment 11

[0076] Based on the clone formation experiment of the intestinal crypt cell line IEC-6 irradiated with γ-rays, the effect of purified oligosaccharides from Trillium japonicum on its clone formation ability was observed.

[0077] 1. Preparation of test substances

[0078] a. Preparation of purified oligosaccharides from Trillium:

[0079] 6 kg of Trillium rhizome was crushed into coarse powder, soaked in 50% ethanol for 1 hour, and extracted by heating and refluxing with 10 times the amount of solvent for 3 times, 2 hours each time, filtered, and the filtrate was combined. The above filtrate was concentrated under reduced pressure at 60-70 ° C to 1.0 g of raw medicinal material / ml, refrigerated at 0-4 ° C overnight, filtered, and the filtrate was enriched on SP825 macroporous adsorption resin column, eluted with 15% ethanol to obtain the eluate. The eluate was concentrated under reduced pressure to a small volume, and the concentrate was again applied to HP20 macroporous adsorption resin, eluted with water and 15% ethanol respectively, and the resin eluate was concentrated and dried with a freeze dryer, and the water eluate and 15% ethanol eluate freeze-dried powder were collected respectively, which was the Trillium purified oligosaccharide, which was recorded as TT-2-0 and TT-2-15 respectively. The above Trillium purified oligosaccharide was prepared into an aqueous solution of appropriate concentration for standby use.

[0080] b. Experimental materials and instruments

[0081] IEC-6 cells are rat intestinal crypt cell lines; clean bench, centrifuge, CO2 constant temperature cell culture box, optical microscope (Olympus), 60 Co ray source, cell viability analyzer, etc.

[0082] c. Experimental methods

[0083] Take out the T75 cell culture flask containing IEC-6 from the CO2 constant temperature cell culture incubator, add 5mL of PBS to wash the cells, and discard the PBS. Add 2mL of 0.25% Trypsin-EDTA to digest the cells for about 3 minutes. Under the microscope, it was observed that most of the cells had been digested. Add 8mL of cell culture medium and mix well; aspirate all the liquid in the cell culture flask and transfer it to a 15mL centrifuge tube, centrifuge at 1000rpm for 5 minutes, discard the supernatant, add 1mL of cell culture medium to the cell pellet, and resuspend the cell pellet. Use 60IEC-6 cells were irradiated with a Co ray source (γ-ray) at a dose of 10 Gy and a dose rate of 54.22 cGy / min. After irradiation, the cells were centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, 1 mL of cell culture medium was added, the cell pellet was resuspended, and the cell viability was counted in a cell viability analyzer. A 6-well plate was taken and the cell density was 1×10 3 Calculate the number of cells required for inoculation based on the number of cells / mL. Inoculate the cells in a 6-well plate. Set up a control group and an experimental group at the same time. Mix the cells and place them in a CO2 constant temperature cell culture incubator for culture. Change the medium every day. After 7 days of culture, remove the crystal violet staining solution and return to room temperature. Remove the 6-well plate, discard the old culture medium in the well, add 1mL of PBS to wash the cells, and discard PBS. Add 1mL of crystal violet staining solution and let it stand at room temperature for 30 minutes. Discard the crystal violet staining solution, add 1mL of H2O to wash the cells, discard H2O, dry the 6-well plate in a fume hood, and then observe and count the number of cell clones under a gel imager.

[0084] d. Experimental results

[0085] The experimental results show (such as Figure 1 As shown in the figure, compared with the control group (without the addition of purified oligosaccharides from Trillium), purified oligosaccharides from Trillium (TT-2-0) and (TT-2-15) significantly increased the number of clones formed by IEC-6 cells irradiated with gamma rays. This indicates that purified oligosaccharides from Trillium TT-2-0 and TT-2-15 have the ability to enhance the clone formation ability of IEC-6 cells irradiated with gamma rays.

[0086] The above results indicate that purified oligosaccharides from Trillium may have the ability to promote the proliferation of intestinal crypt cells after irradiation.

[0087] Example 12

[0088] 1. Preparation of test substances

[0089] Preparation of oligosaccharide fractions from Trillium

[0090] 6 kg of Trillium rhizome was crushed into coarse powder, soaked in 50% ethanol for 1 hour, and extracted by heating and refluxing with 10 times the amount of solvent for 3 times, 2 hours each time, filtered, and the filtrate was combined. The above filtrate was concentrated under reduced pressure at 60-70°C to 1.0 g of raw medicinal material / ml, refrigerated at 0-4°C overnight, filtered, and the filtrate was enriched on an AB-8 macroporous adsorption resin column, eluted with 15% ethanol, and an eluate was obtained. The eluate was concentrated under reduced pressure, the concentrate was dried with a freeze dryer, and the freeze-dried powder was collected, which was the Trillium oligosaccharide part.

[0091] The above oligosaccharide parts of Trillium are prepared into an aqueous solution of appropriate concentration for later use.

[0092] 2. Experimental Materials and Instruments

[0093] IEC-6 cells are rat intestinal crypt cell lines; clean bench, centrifuge, CO2 constant temperature cell culture incubator, optical microscope, 60 Co ray source, cell viability analyzer, confocal microscope, flow cytometer, etc.

[0094] 3. Experimental Methods

[0095] 3.1 IEC-6 cell apoptosis detection

[0096] Take out the T75 cell culture flask containing IEC-6 from the CO2 constant temperature cell culture incubator, add 5mL of PBS to wash the cells, and discard the PBS. Add 2mL of 0.25% Trypsin-EDTA to digest the cells for about 3 minutes. Under the microscope, it was observed that most of the cells had been digested. Add 8mL of cell culture medium and mix well; aspirate all the liquid in the cell culture flask and transfer it to a 15mL centrifuge tube, centrifuge at 1000rpm for 5 minutes, discard the supernatant, add 1mL of cell culture medium to the cell pellet, and resuspend the cell pellet. Use 60 IEC-6 cells were irradiated with a Co ray source (γ-ray) at a dose of 10 Gy and a dose rate of 54.22 cGy / min. After irradiation, the cells were centrifuged at 1000 rpm for 5 minutes and a T25 cell culture flask was taken out and the cell density was 4 × 10 6 Cells / mL were used to calculate the required cell volume for inoculation, and the cells were inoculated in a T25 cell culture flask. A control group and an experimental group were set up at the same time. After mixing the cells, they were placed in a CO2 constant temperature cell culture incubator for culture. After 48 hours of culture, the cells were digested and collected, and the old culture medium was recovered and centrifuged at 1000rpm for 5 minutes. After centrifugation, the supernatant was discarded, 1mL of PBS was added, the cell pellet was resuspended, centrifuged at 1000rpm for 5 minutes, and counted in a cell viability analyzer. Dilute 10×Annexin V Binding Solution to 1×Annexin VBinding Solution with distilled water. Take out the centrifuge tube, discard the supernatant, and add 1×Annexin V Binding Solution to make a final concentration of 1×10 6 Cell suspension of 100 μL cells / mL is recommended. It is recommended not to exceed 500 μL of cell suspension. Control groups are set up: unstained cells, Annexin V and FITC stained cells (without PI), and PI stained cells (without Annexin V and FITC). Take 100 μL of cell suspension and add it to a new 1.5 mL EP tube. Add 5 μL of Annexin V and FITC conjugate, and then add 5 μL of PI solution, and incubate at room temperature in the dark for 15 minutes. Add 400 μL of 1× Annexin V Binding Solution and detect cell apoptosis on a flow cytometer within 1 hour.

[0097] 3.2 EdU flow cytometry detection

[0098] Add EdU (10 μM, reagent A) 2 hours in advance and incubate, then digest and collect the cells; add 3 mL of 1% BSA in PBS to wash the cells, centrifuge at 2000 rpm for 5 minutes, and discard the supernatant. Add 100 μL of Resuspend the cells with fixative (reagent D), mix well and incubate at room temperature in the dark for 15 minutes. Add 3 mL of PBS containing 1% BSA to wash the cells, centrifuge at 2000 rpm for 5 minutes, and discard the supernatant. Add 100 μL of Resuspend the cells in saponin-based permeabilization and wash reagent (reagent E), mix well, and incubate at room temperature in the dark for 15 minutes. EdU bufferadditive (reagent G) was prepared at a ratio of 1:9 Add 0.5 mL of EdU buffer to the roots. Plus reaction cocktails, pipette to mix the cells, and incubate at room temperature in the dark for 30 minutes; add 3 mL of Wash the cells with saponin-based permeabilization and wash reagent (reagent E), centrifuge at 2000 rpm for 5 minutes, and discard the supernatant. Resuspend the cells with 1 mL of PBS, centrifuge at 2000 rpm for 5 minutes, discard the supernatant, resuspend with 250 μL of PBS, filter the cells into a 96-well plate using a 70 μm cell sieve, and detect the EdU incorporation in the cells using a flow cytometer.

[0099] 4. Experimental Results

[0100] The experimental results show (such as Figure 2 As shown in the figure, compared with the control group (without the addition of Trillium oligosaccharide fraction), the percentage of apoptotic cells in the IEC-6 cells irradiated with gamma rays was significantly reduced in the Trillium oligosaccharide fraction (TT-2) group (**p<0.01). The above results show that Trillium oligosaccharide fraction (TT-2) can inhibit the apoptosis of IEC-6 cells irradiated with gamma rays.

[0101] The experimental results show that ( Figure 3As shown in Figure 2, compared with the control group (no addition of Trillium oligosaccharide fraction, 4.25±0.250%), the Trillium oligosaccharide fraction (TT-2) group significantly enhanced the EdU incorporation percentage in the nucleus of γ-ray irradiated IEC-6 cells (**p<0.01). The above results show that Trillium oligosaccharide fraction (TT-2) can promote the proliferation of γ-ray irradiated IEC-6 cells.

[0102] The present invention adopts a γ-ray irradiation radiation injury mouse model, an X-ray irradiation radiation injury mouse model, and a γ-ray irradiated rat small intestinal crypt cell line IEC-6 cell clone formation experiment to observe the effect of Trillium extract on the survival rate of irradiated mice, the effect of Trillium oligosaccharide part (TT-2) and its purified oligosaccharides (TT-2-0 and TT-2-15) on the clone formation ability of rat small intestinal crypt cell line IEC-6 cells, and the effect of Trillium oligosaccharide part (TT-2) on the proliferation and apoptosis of irradiated IEC-6 cells. The results show that the Trillium extract can significantly improve the survival rate of radiation-damaged mice irradiated with γ-rays, X-rays, etc.; the Trillium oligosaccharide part (TT-2) and its purified oligosaccharides (TT-2-0 and TT-2-15) have a significant protective effect on the intestinal damage of rats irradiated with γ-rays, which provides a theoretical basis for further developing radiation damage protection products, especially new natural radiation protection agents for intestinal tissue radiation damage.

[0103] The process parameters (such as temperature, time, etc.) of the present invention can realize the method by taking upper and lower limits and interval values, and the embodiments are not listed one by one here.

[0104] Any contents not described in detail in the present invention can be based on the conventional technical knowledge in the art.

[0105] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0106] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. The use of Trillium extract in the preparation of anti-radiation damage products, characterized in that: The preparation method of the Trillium extract comprises: Step 1: crush the rhizome of Trillium to obtain Trillium powder; Step 2: soaking the Trillium powder in a first ethanol solution, decocting or refluxing for extraction, filtering, collecting the filtrate, concentrating and drying to obtain the Trillium extract, wherein the concentration of the first ethanol solution is 0-50% by volume; The radiation damage is ionizing radiation damage; The ionizing radiation is gamma rays or X-rays.

2. The use of Trillium extract in the preparation of radiation damage resistance products, characterized in that: The preparation method of the Trillium extract comprises: Step 1: crush the rhizome of Trillium to obtain Trillium powder; Step 2: soaking the Trillium powder in a first ethanol solution, decocting or refluxing for extraction, filtering, collecting the filtrate, concentrating and drying to obtain the Trillium extract, wherein the concentration of the first ethanol solution is 0-50% by volume; Before the concentration and drying, the filtrate is further subjected to the following operation: the filtrate is added to a chromatography column, eluted with a second ethanol solution, and the effluent is collected for the concentration and drying to obtain the Trillium extract, wherein the concentration of the second ethanol solution is 0-20% by volume; The chromatography column is filled with a macroporous adsorption resin, and the macroporous adsorption resin is AB-8 type, D101 type, SP825 type or HP20 type; The radiation damage is intestinal damage caused by gamma ray irradiation.

3. The use according to claim 2, characterized in that: Before the concentration and drying, the effluent is added to the chromatography column again, eluted with a third ethanol solution, and the effluent is collected for concentration and drying to obtain the Trillium extract, wherein the concentration of the third ethanol solution is 0-20% by volume.

4. The use according to claim 1 or 2, characterized in that: The medicinal material source of the Trillium rhizome is a plant of the genus Trillium in the Liliaceae family, including but not limited to Trillium Trillium tschonoskii Maxim., Trillium jilinense Trillium kamtschaticum Pall. ex Pursh and Tibetan Trillium Trillium govanianum One or more of Wall. ex Royle.

5. The use according to claim 1 or 2, characterized in that: The anti-radiation damage products include auxiliary anti-radiation damage health foods.

6. The use according to claim 1 or 2, characterized in that: The anti-radiation damage products include drugs.

7. The use according to claim 6, characterized in that: The dosage form of the medicine is granules, oral solution, capsule, tablet or injection.

8. The use according to claim 6, characterized in that: The dosage form of the medicine is powder injection.

9. The use according to claim 6, characterized in that: The dosage form of the medicine is water injection.

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