Method for synergistically extracting polysaccharides from kuding tea and radiation protection application

Abstract

The present application provides a method for synergistically extracting polysaccharides from Kuding tea and an application of the polysaccharides in radiation protection. The extraction process of the polysaccharides comprises the following steps: subjecting the dried Kuding tea to steam explosion treatment, crushing the Kuding tea into particles with a size of 90-100 mesh, immersing the Kuding tea particles in distilled water, adding the solution into a microwave countercurrent extraction device, and simultaneously applying ultrahigh pressure for synergistic extraction to obtain an extraction solution. After centrifugation, the supernatant is collected, Sevage solution is added to remove proteins, and the supernatant is collected. Then, anhydrous ethanol is added to the supernatant, followed by centrifugation and freeze-drying to obtain the polysaccharides. The present application provides a method for synergistically extracting polysaccharides from Kuding tea, and it is found that the polysaccharides have a radiation protection effect, which provides a basis for further deep processing and a preparation method with a higher extraction rate.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical chemistry, and particularly relates to a method for synergistically extracting polysaccharides from Ilicium religiosum and radiation application. BACKGROUND

[0002] With the development of science and technology and the increase of human activities, the influence of ionizing radiation on human health and the environment is becoming increasingly large. Ionizing radiation mainly includes electromagnetic radiation and particle radiation. Electromagnetic radiation includes visible light, ultraviolet light, X-rays and gamma rays, while particle radiation includes alpha particles, beta particles and neutrons.

[0003] The influence of ionizing radiation on the human body mainly manifests in the cellular and tissue levels. When ionizing radiation interacts with human cells, a series of biological effects are produced. Among them, the direct effect refers to the interaction of ionizing radiation with nucleic acids, proteins and other important biological molecules in the cell nucleus, leading to DNA breakage, protein structure change, etc. The indirect effect refers to the interaction of ionizing radiation with water molecules in the cell, producing reactive oxygen free radicals, and then triggering cell damage and oxidative stress response. The influence of ionizing radiation on the environment cannot be ignored. It can lead to the accumulation of radioactive substances in the soil and water, and then affect the stability of the biological chain and the balance of the ecological system. In addition, ionizing radiation also has negative effects on plant growth and development, leading to reduced crop yields and reduced biodiversity.

[0004] Ilicium religiosum is a traditional Chinese medicinal material, mainly processed from the leaves or branches of plants in the genus Litsea of the family Lauraceae. Due to its rich medicinal ingredients, Ilicium religiosum has a wide range of applications in the field of traditional Chinese medicine. Studies have shown that Ilicium religiosum is rich in volatile oils, flavonoids, monoterpenes and other active ingredients. Among them, volatile oils are one of the main components of Ilicium religiosum, and have antibacterial, anti-inflammatory and antioxidant activities. Flavonoids have antioxidant, anti-tumor and anti-inflammatory effects, and play an important protective role in human health. Among them, Ilicium religiosum polysaccharides are a natural bioactive substance, which has antioxidant, immunomodulatory, anti-tumor, hypoglycemic, hypolipidemic, liver-protective and other important biological functions and pharmacological effects. Further research and development of Ilicium religiosum polysaccharides will provide new strategies for human radiation protection. SUMMARY

[0005] In order to overcome the shortcomings of traditional Ilicium religiosum polysaccharide extraction, such as high temperature, long time consumption and large amount of extraction solvent, the present application provides a method for synergistically extracting Ilicium religiosum polysaccharides by using microwave countercurrent and ultra-high pressure. It is found that the extracted Ilicium religiosum polysaccharides can be used for the preparation of anti-radiation drugs.

[0006] To achieve the above object, one of the purposes of the present application adopts the technical solution: a method for synergistically extracting Ilicis Latifoliae Polysaccharide and radiation protection application, characterized in that the method steps are:

[0007] Step 1) washing the Ilicis Latifoliae leaves, drying the Ilicis Latifoliae in a 70°C oven for 48 h;

[0008] Step 2) steam explosion treatment: steam explosion treatment is performed on the Ilicis Latifoliae, the pressure is 1.6 Mpa, the pressure holding time is 2 min, and then a pulverizer is used to pulverize the Ilicis Latifoliae into 90-100 mesh Ilicis Latifoliae particles;

[0009] Step 3) weighing the Ilicis Latifoliae dry powder, and soaking the above Ilicis Latifoliae particles in distilled water;

[0010] Step 4) adding the solution to a microwave countercurrent extraction device for microwave extraction, and applying ultrahigh pressure in the process, repeating the extraction operation 4 times, each time for 10 minutes, to obtain an extraction solution, wherein the microwave is a penetrating heating type;

[0011] Step 5) centrifuging the extracted solution at a speed of 10000 r / min for 15 min, and collecting the supernatant;

[0012] Step 6) mixing the supernatant with Sevage solution at a volume ratio of 3:1 to remove protein, collecting the supernatant 3 times;

[0013] Step 7) after combining the three supernatants, rotary evaporation is performed to concentrate to 1 / 3 of the volume, then 4 times the volume of anhydrous ethanol is added, sealed and left overnight, centrifuged to obtain the precipitate, and the precipitate is freeze-dried to obtain Ilicis Latifoliae polysaccharide powder.

[0014] Further, the extraction pressure of step 4) is 400-500 MPa, the extraction time is 10 min, the solid-liquid ratio of Ilicis Latifoliae dry powder to distilled water is 1:20-50 g / m, and the microwave power of the microwave countercurrent extraction device is 1000 WL.

[0015] Further, the Sevage solution in step 6) is a mixture of chloroform and n-butanol at a volume ratio of 4:1.

[0016] The second purpose of the present application is to provide the use of Ilicis Latifoliae polysaccharide in the preparation of radiation protection drugs:

[0017] The working principle and beneficial effects of the present application are as follows: taking Ilicium fortunei as raw material, microwave countercurrent and ultrahigh pressure are used to extract polysaccharide, and Ilicium fortunei polysaccharide is obtained after freeze-drying. Ilicium fortunei polysaccharide can be metabolized by intestinal flora to produce a series of metabolites, including short-chain fatty acids (such as propionic acid, butyric acid and acetic acid), polypeptides, antioxidant substances and the like. These metabolites can affect the function and behavior of the central nervous system through the gut-brain axis, such as neurotransmitter synthesis, immune response and metabolite signal transmission, thereby playing a radiation protection role.

[0018] Compared with the prior art, the present application has the following advantages and effects:

[0019] The present application uses microwave countercurrent and ultrahigh pressure to extract, which has the advantages of low extraction temperature, short extraction time and high extraction rate.

[0020] 2) The present application has a wide range of applications, as the influence of ionizing radiation on human health and the environment is becoming more and more significant, and Ilicium fortunei polysaccharide can play a protective role, thus having a broad application market. DETAILED DESCRIPTION

[0021] The present application will be further described in detail through specific embodiments as follows:

[0022] 1. Preparation of Ilicium fortunei polysaccharide

[0023] In order to make the purpose, technical scheme and advantages of the present application more clear and explicit, the present application will be further described in detail in combination with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and not to limit the present application.

[0024] The present embodiment provides a method for extracting Ilicium fortunei polysaccharide by microwave countercurrent and ultrahigh pressure, and the method steps are as follows:

[0025] Step 1) The Ilicium fortunei leaves are cleaned, and the Ilicium fortunei is dried in a 70℃ oven for 48 h;

[0026] Step 2) Steam explosion treatment: the Ilicium fortunei is subjected to steam explosion treatment at a pressure of 1.6 Mpa for 2 min, and then is crushed into Ilicium fortunei particles of 90-100 mesh by a crusher;

[0027] Step 3) Ilicium fortunei dry powder is weighed, and the above Ilicium fortunei particles are soaked in distilled water;

[0028] Step 4) The solution is added to a microwave countercurrent extraction device for microwave extraction, and ultrahigh pressure is applied for synergistic extraction during the process. The extraction is repeated 4 times, and each extraction time is 10 minutes to obtain an extraction liquid. The microwave is a penetrating heating type.

[0029] Step 5) centrifuge the extracted solution at 10,000 r / min for 15 min, and collect the supernatant;

[0030] Step 6) mix the supernatant with Sevage solution at a volume ratio of 3:1, remove the protein, collect the supernatant, and collect three times;

[0031] Step 7) after combining the three supernatants, rotary evaporation is performed to concentrate to 1 / 3 of the volume, then 4 times the volume of anhydrous ethanol is added, sealed and placed overnight, centrifuged to obtain the precipitate, and the precipitate is freeze-dried to obtain the Ilex kudingcha polysaccharide powder.

[0032] The phenol-sulfuric acid method is used to determine the content of the Ilex kudingcha polysaccharide.

[0033] 2, monosaccharide composition analysis of Ilex kudingcha polysaccharide HTP

[0034] HPLC conditions: the mobile phase is composed of acetonitrile: phosphate buffer salt = 18.2:81.8. Detector model: Elitte EClassical 3200, detection wavelength: 254 nm. Chromatographic column model: Supersil ODS2 (4.6*250mm); column temperature: 35℃. Injection volume: 20μL, flow rate: 1mL / min.

[0035] Sample pretreatment:

[0036] (1) acid hydrolysis: weigh 2mg of Ilex kudingcha polysaccharide sample into a brown vial, dissolve it in 2mL of 4M trifluoroacetic acid (TFA), seal and heat hydrolysis at 110℃ for 4h, then cool to room temperature, use methanol to concentrate under reduced pressure, and repeat the operation until all TFA is removed. After evaporation, dissolve the residue in 200μL of distilled water.

[0037] (2) PMP derivatization: take 100μL of completely acid hydrolysis solution in (1), add 100μL of 0.6M NaOH solution, mix, then add 200μL of freshly prepared 0.5M PMP methanol solution, seal and vortex mix. React at 70℃ for 100min, then cool to room temperature, neutralize with 200μL of 0.3M HCl, and make up to 1mL with distilled water. Add 1mL of chloroform for extraction, take out the upper aqueous phase, and repeat the extraction several times to remove as much PMP as possible. After vortexing, take 20μL of supernatant for analysis.

[0038] (3) take 100μL of 1mg / mL monosaccharide standard mixture (Man, Rha, Gal, GalA, Glc, GlcA, Xyl, Ara, Fuc), and process it in the same way as the sample PMP derivatization, then perform HPLC analysis.

[0039] The purified polysaccharide of I. latifolia is mainly composed of five monosaccharides of rhamnose, arabinose, galactose, glucose and mannose, with percentages of 9.50%, 46.87%, 38.03%, 4.60% and 1.00%, respectively.

[0040] Radiation resistance activity determination of I. latifolia polysaccharide

[0041] The cells used in the experiment were BV2 cells purchased from Beijing Union Hospital. The cell culture medium used was 90% DMEM + 10% fetal bovine serum. The culture was carried out in a 37°C, 0.5% CO2 incubator.

[0042] According to the experimental requirements, the cells were divided into Control group, radiation group: 8 Gy radiation group, 16 Gy radiation group, 32 Gy radiation group, 64 Gy radiation group; Acanthopanax senticosus extraction intervention group: 50 μg / mL, 100 μg / mL, 250 μg / mL, 500 μg / mL, 1000 μg / mL; Radiation + Acanthopanax intervention group: 32 Gy + 250 μg / mL, 32 Gy + 500 μg / mL, 32 Gy + 1000 μg / mL; and positive control group (donepezil hydrochloride): 32 Gy + donepezil. Among them, the Control group was not treated with irradiation, and serum-free DMEM medium was added as a control. The radiation group was given different doses (8 Gy, 16 Gy, 32 Gy, 64 Gy) of 60CO-γ ray irradiation, and serum-free DMEM medium was added as a control. The I. latifolia polysaccharide intervention group was treated with different concentrations of serum-free DMEM medium prepared I. latifolia polysaccharide (50 μg / mL, 100 μg / mL, 250 μg / mL, 500 μg / mL, 1000 μg / mL) for 24 h. The radiation + Acanthopanax intervention group used different concentrations of I. latifolia polysaccharide (250 μg / mL, 500 μg / mL, 1000 μg / mL)

[0043] After 24 h of treatment, 60CO-γ ray irradiation was performed at a total dose of 32 Gy. The positive control group was treated with donepezil hydrochloride at a final concentration of 100 μmol / L for 24 h, and then 60CO-γ ray irradiation was performed at a total dose of 32 Gy. The radiation dose rate used in the experiment was 1 Gy / min.

[0044] Radiation treatment method: BV2 cells were seeded in 6-well plates at a density of 1 x 104 / well, and after the cells adhered, according to the grouping conditions, the polysaccharides of Kuding tea or serum-free DMEM medium were added for 24 h, then the well plate was sealed to avoid bacterial contamination. The well plate was placed under a 60CO-γ radiation source, and irradiated at 1 Gy / min to a total dose of 8 Gy, 16 Gy, 32 Gy, and 64 Gy, respectively. After 12 h of radiation treatment, the cells and supernatant were collected and stored at -80°C for testing.

[0045] Stimulation of BV2 cells with different concentrations of Kuding tea polysaccharides (0-1000 μg / mL) did not cause a decrease in cell viability, and 24 h before radiation stimulation, the cells were treated with positive drugs and Kuding tea polysaccharides, respectively. The results showed that Kuding tea polysaccharide intervention 24 h in advance can prevent the decrease in cell viability caused by irradiation, and Kuding tea polysaccharides can be developed into anti-ionizing radiation drugs.

[0046] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for synergistically extracting polysaccharides from Kuding tea, characterized in that: The method steps are: Step 1) steam explosion treatment: the dried kuding tea is subjected to steam explosion treatment, the pressure is 1.6 Mpa, the pressure holding time is 2 min, and then it is crushed into kuding tea particles with a size of 90-100 mesh; Step 2) the kuding tea particles are soaked in distilled water, the solution is added to a microwave countercurrent extraction device, and superhigh pressure is applied for synergistic extraction, the extraction operation is repeated 4 times, each time for 10 min, and an extraction solution is obtained; Step 3) the extraction solution is centrifuged at a speed of 10000 r / min for 15 min, the supernatant is collected, then Sevage solution with a volume of three times that of the supernatant is added to remove proteins, the supernatant is collected, and the collection is repeated 3 times; Step 4) the supernatant is concentrated, 4 times the volume of anhydrous ethanol is added, centrifuged, and freeze-dried to obtain kuding tea polysaccharides; The extraction pressure is 400-500 MPa, the extraction time is 10 min, and the microwave power is 1000 W.

2. The use of kuding tea polysaccharides in the preparation of radiation protection drugs according to claim 1.