CN113908180B

CN113908180B - Application of nano-selenium cordyceps militaris aqueous extract in reducing radiotherapy injury and protective agent thereof

Info

Publication number: CN113908180B
Application number: CN202111343688.XA
Authority: CN
Inventors: 陈填烽; 刘畅; 李海伟; 陈义康; 李绿漪; 陈雯婷
Original assignee: Guangdong Jicheng Selenium Source Nano Research Institute Co ltd
Current assignee: Guangdong Jicheng Selenium Source Nano Research Institute Co ltd
Priority date: 2021-11-13
Filing date: 2021-11-13
Publication date: 2022-12-16
Anticipated expiration: 2041-11-13
Also published as: CN113908180A

Abstract

The invention discloses application of a nano-selenium cordyceps militaris aqueous extract in reducing radiotherapy injury and a protective agent thereof.

Description

Application of nano-selenium cordyceps militaris aqueous extract in reducing radiotherapy injury and protective agent thereof

Technical Field

The patent relates to the technical field of compound medicines, in particular to application of nano-selenium cordyceps militaris aqueous extract in reducing radiotherapy injury and a protective agent thereof.

Background

Selenium (Se) has an important effect on human health, is a necessary trace element for human, is praised as ' fire of life ' by the medical and nutritional communities at home and abroad, and enjoys the reputations of ' long life element ', ' king of cancer resistance ', ' heart guard and the like. The biological functions of the medicine are as follows: improving immunity, resisting oxidation, delaying aging, protecting and repairing cell, preventing and resisting cancer, removing toxic substance from heavy metal, and protecting eyes. Therefore, the reasonable intake of selenium element has important effects on human health and disease prevention. However, more than two thirds of areas lack of selenium exist in China, and the number of the areas lack of selenium is up to 22. Selenium deficiency seriously threatens the national peopleThe health of the human body is closely related to keshan disease, kaschin-Beck disease and the like. The Chinese Nutrition society and the FA0/WHO/IAEA joint expert Committee determine that the suitable range of the human intake is 60-250 mug/d, the safe dose is 400 mug/d, and the toxic dose is 800 mug/d. Excessive selenium can also cause harm to human bodies, so that a safe and efficient selenium source needs to be developed for people with selenium deficiency. Selenium mainly exists in two forms of organic selenium and inorganic selenium in nature, usually the inorganic selenium (selenate, selenite and the like) has high toxicity and narrow safety window, and has potential danger when being directly used as a selenium supplement source; and the organic selenium (selenomethionine, selenocysteine, selenium yeast and the like) has low toxicity and has various biological activities. In acute toxicity (LD) ₅₀ ) In one aspect, the inorganic selenium is 15 mg/kg, the organic selenium is 35 mg/kg, and the nano-selenium is 113 mg/kg. Therefore, the biological conversion of inorganic selenium into organic selenium with high activity is one of the most safe and effective means for selenium products at present. With the development of nanotechnology, compared with inorganic selenium and organic selenium, elemental selenium (SeNPs) has been widely paid attention to because it has the characteristics of low toxicity, high bioavailability, high reaction yield, and easy surface modification. Because the toxicity of the nano-selenium is low, the absorption and tolerance concentration of the nano-selenium by organisms is also obviously higher than that of inorganic selenium, so that the nano-selenium is safer and more efficient in the biotransformation process. Our previous studies have demonstrated that nano-selenium can reverse cisplatin-induced renal injury and fatty acid-induced islet cell injury.

Cordyceps militaris belongs to genus Cordyceps of Clavipitaceae of order Sphaeriales of class Sclerotinia of phylum Eumycota, subphylum Ascomycotina. Cordycepin, adenosine, cordycepic acid and the like are main components of cordyceps militaris playing roles. Research shows that cordycepin is an important active ingredient of cordyceps militaris, and the cordycepin content in cordyceps militaris is tens of times that of cordyceps militaris, so that the cordycepin is a main source for obtaining cordycepin. Cordyceps militaris has various physiological activities, including antiviral, antiinflammatory, antioxidant, antiaging, antitumor, antiproliferative, radioprotective, nerve protecting, kidney protecting, liver protecting, and immunity enhancing effects. The cordyceps militaris is used as a substitute of wild cordyceps militaris, and the market space is large. The artificial cultivation of the cordyceps militaris has the advantages of low investment, high benefit and short production period, and can be cultivated all the year round. Cordycepin has been shown to have anti-inflammatory, anti-tumor, antioxidant and cytostatic biological activities. Cordycepin can exert an anti-inflammatory effect in lipopolysaccharide-induced macrophage damage in part by inhibiting NLRP3 inflammasome and cyclooxygenase 2 mediated inflammatory responses. Cordycepin plays a protective role in myocardial injury by regulating NF-kB signal channels. Meanwhile, the cordyceps militaris has obvious effects on inhibiting cancer cell proliferation and inducing cancer cell apoptosis. However, the selenium content in the common cordyceps militaris sporocarp is very low, and the selenium content in the cordyceps militaris is greatly improved through the biotransformation of the cordyceps militaris by adding the nano-selenium into the culture medium, and exists in the sporocarp in the form of organic selenium basically, so that the selenium is very easy to be absorbed and utilized by a human body.

The current measures for protecting radiotherapy are mainly used for a protective agent before radiation and a therapeutic agent after radiation, and comprise chemical radioprotectors, such as a protective agent containing compounds, plant and Chinese herbal medicines, cytokines, mesenchymal stem cell radiation therapeutic agents and the like. The drugs approved by the Food and Drug Administration (FDA) for clinical radiotherapy protection are Amifostine (Amifostine) and Palifermin (Palifermin), but these two drugs cause adverse reactions such as hypotension, hypocalcemia, nausea and vomiting, and have various toxic and side effects. Because the half-life period of the amifostine in vivo is shorter, the amifostine has protection effect only when being used immediately before chemotherapy, and the drug response is closely related to the administration time. Therefore, amifostine must be administered 30 min before chemotherapy, with a short effective time window and at high cost.

The inventor has previously applied for a patent with the patent number of ZL201811313225.7, and finds that the nano-selenium cordyceps militaris has the excellent characteristics of cordyceps militaris and selenium through further research on the basis of the culture mode of the patent, can be used as food, has value in medical application and needs to be proved by experiments.

Disclosure of Invention

The invention mainly aims to find that the application of the nano-selenium cordyceps militaris aqueous extract in reducing radiation injury has good effects of removing free radicals and resisting oxidation, has a good protection effect on radioactive injury of a body, and has long effective protection time and small adverse reaction.

The invention also aims to provide the protective agent for the nano-selenium cordyceps militaris aqueous extract, which can reduce the damage of human cells in radiotherapy and particularly has a protective effect on the damage of human cardiac muscle caused by radiotherapy.

The inventor finds that the nano-selenium cordyceps militaris has the excellent characteristics of cordyceps militaris and selenium, is a natural medicament, has good pharmacological activity, more importantly, has good effect of antagonizing radiotherapy injury, has basically negligible toxicity compared with a common protective agent, and is a novel protective agent worthy of research and development. Therefore, the nano-selenium cordyceps militaris has protective activity on myocardial damage caused by radiotherapy, and provides a new scientific basis for developing a high-efficiency and safe radiotherapy protective agent.

The invention provides an application of a nano-selenium cordyceps militaris aqueous extract in reducing radiation-treatment damage.

In particular to the application of reducing the radiation therapy injury to human organs or muscles. Can be used for protecting the injury of cardiac muscle and lung of human body caused by radiotherapy.

Furthermore, the nano-selenium cordyceps militaris aqueous extract has the effects of removing free radicals and resisting oxidation, and reduces the damage of cells in radiotherapy.

Further, the content of adenosine in the nano-selenium cordyceps militaris aqueous extract is 0.21-0.28%, the content of total selenium is 80.17-198.02 mu g/g, and the proportion of selenocysteine in the total selenium is 17.23-49.07%.

The invention also provides a protective agent capable of reducing radiation therapy injury, which comprises the nano-selenium cordyceps militaris water extract. In particular to a nano-selenium cordyceps militaris aqueous extract with high adenosine content.

Further, the content of adenosine in the nano-selenium cordyceps militaris water extract is 0.21-0.28%, the content of total selenium is 80.17-198.02 mu g/g, and the content of selenocysteine in the total selenium is 17.23-49.07%.

Further, the protective agent may be administered orally.

Compared with the prior art, the scheme of the invention has the following advantages and effects:

(1) A large number of experiments prove that the nano-selenium cordyceps militaris aqueous extract has the strongest oxidation resistance under the same selenium content. Specifically, at the in vitro level, the inventor compares the scavenging capacity of selenium in different forms such as sodium selenite, sodium selenate, nano-selenium, selenocysteine, selenomethionine, methyl selenocysteine, nano-selenium cordyceps militaris aqueous extract and the like on ROS caused by radiotherapy, and the result shows that the nano-selenium cordyceps militaris aqueous extract has the strongest scavenging capacity, and the scavenging capacity is improved by 20-90%. The inventor also considers the scavenging capacity of the aqueous extract of cordyceps militaris and the aqueous extract of nano-selenium cordyceps militaris on ROS caused by radiotherapy, and finds that the scavenging capacity of the aqueous extract of nano-selenium cordyceps militaris is stronger than that of the aqueous extract of cordyceps militaris, and the scavenging capacity of the aqueous extract of nano-selenium cordyceps militaris is 1.3-2.1 times higher than that of the aqueous extract of cordyceps militaris.

(2) Through a large number of experiments, the inventor discovers the application of the nano-selenium cordyceps militaris water extract with high adenosine content in reducing radiation treatment injury. The radiation-proof health-care product has a certain radiation-proof effect, low toxicity, very strong cell repairing effect and a good protection effect on radioactive damage to a human body.

The nano-selenium cordyceps militaris water extract has high selenium content, good free radical removal and oxidation resistance effects, and good protection effect on myocardial damage caused by radiotherapy.

The invention greatly expands the application field of nano-selenium cordyceps militaris and has wide medical drug application prospect. The raw materials of the protective agent are cheap and easy to obtain, the synthetic purification steps have strong operability, the synthetic scale is easy to expand, and the commercialization and the application of the protective agent medicine are realized.

Drawings

FIG. 1: is the toxicity of different radiotherapy doses to H9C2 cells.

FIG. 2: the results are photographs of H9C2 cells at a radiation dose of 8Gy and the cell viability.

FIG. 3: the nano-selenium cordyceps militaris water extract reduces the increase of ROS caused by radiotherapy.

FIG. 4: is a graph showing changes in mitochondrial membrane potential of H9C2 cells.

FIG. 5 is a schematic view of: is a graph of the change in body weight of mice.

FIG. 6: is the proportion change of macrophages in the mouse spleen.

FIG. 7: is a graph showing the changes of Mn-SOD and cTn in mouse serum.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

The invention provides application of a nano-selenium cordyceps militaris aqueous extract in reducing radiotherapy damage, and particularly relates to application of a nano-selenium cordyceps militaris aqueous extract with high adenosine content in reducing radiotherapy damage. The nano-selenium cordyceps militaris aqueous extract has the effects of removing free radicals and resisting oxidation, and reduces the damage of cells in radiotherapy. For example, the protective effect can be achieved on the myocardial damage of the human body caused by radiotherapy.

The protective agent capable of reducing the radiation damage is obtained by adopting the following method steps:

(1) Inoculating the cultured cordyceps militaris mycelia into a fermentation culture medium for culturing to obtain cordyceps militaris seed liquid;

(2) Adding the polysaccharide composite nano-selenium and the cordyceps militaris seed solution prepared in the step (1) into a cordyceps militaris fruiting body culture medium for continuous culture to obtain nano-selenium cordyceps militaris fruiting bodies;

(3) Weighing a certain amount of the nano-selenium cordyceps militaris sporocarp, adding water after crushing, performing ultrasonic extraction to obtain a nano-selenium cordyceps militaris aqueous extract, and freeze-drying or spray-drying the nano-selenium cordyceps militaris aqueous extract and then storing.

The content of adenosine in the nano-selenium cordyceps militaris water extract is 0.21-0.28%, the content of total selenium is 80.17-198.02 mu g/g, and the proportion of selenocysteine in the total selenium is 17.23-49.07%.

Experiments prove that the nano-selenium cordyceps militaris aqueous extract provided by the invention has the effect of reducing radiation injury. Especially has protective effect on the myocardial damage of human body caused by radiotherapy.

(I) selenium with different forms has the ability of resisting and eliminating free radicals

DPPH stock (dissolved in methanol) was diluted to OD with PBS _{517 nm} And (3) a working solution of = 0.35 ± 0.02. Mixing DPPH working solution (180 μ L) with 20 μ L of sodium selenite, sodium selenate, selenocysteine and Cordyceps militaris water extract with prepared concentration, nano-selenium Cordyceps militaris water extract aqueous solution and positive control Trolox, monitoring OD values at different time under a fluorescence enzyme labeling instrument, and continuously reading for 30 min. The results show that when the concentration of selenium is 0.05 mug/mL, the clearance rates of the nano-selenium cordyceps militaris aqueous extract are respectively improved by 37%, 69% and 21% compared with sodium selenite, sodium selenate and selenocysteine. 0.05 Compared with the Cordyceps militaris aqueous extract of 0.05 mg/mL, the nano-selenium Cordyceps militaris aqueous extract of mg/mL has the capability of removing DPPH free radicals improved by 1.73 times.

(II) influence of different doses of X-ray on myocardial cell survival.

The cell concentration was 3X 10 ⁴ Adding myocardial cells (H9C 2) per mL to 96-well culture plate, adding 100 μ L per well, culturing for 24 hr, irradiating with X-ray at 37 deg.C and 5% CO at different doses ₂ After 72 h incubation, pictures were taken under the microscope and the cell photographs at 8Gy dose are shown in FIG. 2A. After photographing, 20 mu L/well of MTT (5 mg/mL) is added, after 4 h, the supernatant is discarded, 100 mu L/well of DMSO is added, oscillation is carried out for about 10 min, and OD is measured by a microplate reader ₅₇₀ The value is obtained. FIG. 1 is the toxicity of different radiotherapy doses on H9C2 cells.

The survival rate of the cells is calculated and plotted as fig. 1, and it is obvious that under 2 Gy and 4 Gy, the survival rate of the cardiomyocytes is still higher, and under 16 Gy, the survival rate of the cardiomyocytes is too low, so that 8Gy is finally selected as the radiotherapy dose for carrying out the subsequent experiment.

And (III) a myocardial cell radiotherapy injury protection experiment.

The cell concentration was 3X 10 ⁴ Adding each myocardial cell/mL into a 96-well culture plate, adding 100 μ L per well, culturing for 24 h, adding 0.0977-200 μ g/mL Cordyceps militaris water extract and 100 μ L nano-selenium Cordyceps militaris water extract, treating for different time, selecting 8Gy X-ray radiation according to the previous experiment result, placing at 37 deg.C with 5% CO ₂ Culturing for 72 h, adding MTT (5 mg/mL) 20 μ L/well, centrifuging after 5 h, discarding supernatant, adding DMSO 100 μ L/well, shaking for 10 min, and measuring OD with enzyme-labeling instrument ₅₇₀ Cell viability was calculated and plotted as the image of fig. 2 (B).

After comparing the cytoprotective effects of the drug treatments over different periods of time, the above experiments were repeated with concentrations of 0.781-25 μ g/mL. It can be observed that the protection effect of the nano-selenium cordyceps militaris is optimal and higher than that of the common cordyceps militaris under the drug concentration of 6.25 mug/mL.

(IV) the nano-selenium cordyceps militaris aqueous extract reduces ROS caused by X-Ray.

According to the MTT experimental result, the appropriate radiotherapy dose and the appropriate dosing time are selected, and the H9C2 cells are treated at the speed of 5 multiplied by 10 ⁴ Inoculating the strain/mL of the strain in a 6 cm dish, incubating for 24 h, adding a certain amount of common cordyceps aqueous extract and nano-selenium cordyceps aqueous extract, incubating for 24 h, adding 8Gy of radiation dose, continuing incubating for 48 h, collecting cells, incubating a DCF probe for 30 min, and detecting fluorescence intensity by flow cytometry.

Referring to fig. 3, the result shows that 8Gy of X-Ray can significantly increase intracellular ROS, while the addition of common cordyceps militaris and nano-selenium cordyceps militaris aqueous extract can significantly reduce the increase of ROS caused by radiotherapy, and the effect is better than AMI. Compared with the common cordyceps militaris aqueous extract, the nano-selenium cordyceps militaris aqueous extract can also reduce ROS more obviously.

(V) JC-1 detects changes in mitochondrial membrane potential.

Inoculating H9C2 myocardial cells in logarithmic phase into a 6 cm cell culture dish until the cells grow in an adherent manner for 24 hours, and then adding a medicament to incubate the cells for 12 hours; after the cells were irradiated with X-rays (8 Gy), they were further cultured in an incubator for 48 hours. After collecting the cells, 200. Mu.L of 0.9% NaCl solution containing JC-1 probe (10. Mu.g/mL) was added, and the cells were incubated at 37 ℃ for 30 min to detect changes in mitochondrial membrane potential by flow cytometry.

The results are shown in fig. 4, and the experimental results show that radiotherapy can significantly cause the decrease of the mitochondrial membrane potential of the H9C2 cell, after different drugs are added, AMI and the common cordyceps aqueous extract can slightly inhibit the decrease of the mitochondrial membrane potential of the H9C2 cell, and the nano-selenium cordyceps aqueous extract can significantly inhibit the decrease of the mitochondrial membrane potential of the H9C2 cell.

And (VI) evaluating the in vivo protection effect of the mice.

Normal Balb/c mice are fed with 0.5 mg/kg of common cordyceps militaris aqueous extract and nano-selenium cordyceps militaris aqueous extract every day, 5 Gy of radiation dose is given every week for 4 times of radiotherapy, and the administration is continued for 4 weeks after the radiotherapy is finished. During the period, the body weight and other indexes of the mice are detected. After the experiment is finished, the proportion change of macrophages in the spleen of the mouse is detected, and the changes of SOD and myocardial enzymes in serum are detected.

The results are shown below, and the radiotherapy can cause the weight of the mice to be reduced to a certain degree, while the weight of the mice is reduced after the cordyceps militaris aqueous extract and the nano-selenium cordyceps militaris aqueous extract are administered, and the weight of the mice is recovered faster after the radiotherapy is finished (see fig. 5). After the experiment is finished, the proportion of macrophages in the spleen of a mouse is detected, and the result shows that X-ray can reduce the anti-inflammatory type M2 macrophages in the spleen, and the increase of the proportion of the M2 macrophages in the spleen can be improved by the administration of the common cordyceps militaris water extract, the nano-selenium cordyceps militaris water extract and the AMI, and particularly the increase of the proportion of the nano-selenium cordyceps militaris water extract is more obvious (see figure 6). Furthermore, we examined the changes of Mn-SOD and cardiac muscle calcium enzyme cTn in mouse serum, and the results showed that radiotherapy can cause the increase of Mn-SOD and cTn, and HSe can significantly reduce the increase of Mn-SOD and cTn caused by radiotherapy (see FIG. 7).

The experimental results prove that the nano-selenium cordyceps militaris water extract has a good effect of reducing radiotherapy injury, can specifically reduce the radiotherapy injury to human organs or muscles, can remarkably reduce the increase of ROS (reactive oxygen species) caused by radiotherapy, has low toxicity, has a strong cell repair effect, and particularly has a protection effect on the human myocardial injury caused by radiotherapy.

The water extract of the selenium-rich cordyceps militaris has high selenium content, good effects of removing free radicals and resisting oxidation, and good protection effect on myocardial damage caused by radiotherapy, and compared with the existing common medicaments, the water extract of the selenium-rich cordyceps militaris has the advantages that the effective time window is greatly increased, the adverse reaction is almost negligible, and the price is lower. The invention greatly expands the application field of nano-selenium cordyceps militaris and has wide medical drug application prospect.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. The application of the nano-selenium cordyceps militaris aqueous extract in preparing a radiotherapy injury protective agent is characterized in that the content of adenosine in the nano-selenium cordyceps militaris aqueous extract is 0.21-0.28%, the content of total selenium is 80.17-198.02 mu g/g, wherein the proportion of selenocysteine in the total selenium is 17.23-49.07%, and the radiotherapy injury is myocardial injury caused by radiotherapy.

2. The use of claim 1, wherein the nano-selenium cordyceps militaris aqueous extract has free radical scavenging and antioxidant effects.