阅读说明：本技术 柴达木大肥菇酚酸保护急、慢性缺氧损伤的用途 (Application of chaenomeles speciosa phenolic acid in protection of acute and chronic hypoxia injury ) 是由 焦迎春 陈雅楠 于 2019-11-12 设计创作，主要内容包括：本发明公开了柴达木大肥菇酚酸在制备保护急、慢性缺氧损伤的药物、保健品或食品中的用途,经本发明研究表明,柴达木大肥菇酚酸具有延长常压耐缺氧试验缺氧小鼠的死亡时间；在亚硝酸钠中毒试验延长中毒小鼠的存活时间；在急性脑缺血性缺氧试验中延长急性缺氧小鼠喘息时间的作用；可降低缺氧条件下LDH(乳酸脱氢酶)含量、NO(一氧化碳)含量水平,升高ATP(三磷酸腺苷)含量水平,增强机体低氧损伤的耐受能力,提高组织的急性缺氧损伤的代偿能力,保护组织免受缺氧危害,可以将其作为安全可靠的抗缺氧药物、保健品或食品。(The invention discloses application of chaenomeles speciosa phenolic acid in preparation of medicines, health-care products or foods for protecting acute and chronic hypoxia injury, and researches of the invention show that the chaenomeles speciosa phenolic acid can prolong the death time of a hypoxia mouse in a normal-pressure hypoxia-resistant test; prolonging the survival time of poisoned mice in a sodium nitrite poisoning test; the function of prolonging the wheezing time of an acute hypoxia mouse in an acute cerebral ischemic hypoxia test; can reduce LDH (lactate dehydrogenase) content and NO (carbon monoxide) content under the anoxic condition, increase ATP (adenosine triphosphate) content, enhance the tolerance capacity of hypoxia injury of organisms, improve the compensation capacity of acute hypoxia injury of tissues, protect the tissues from being damaged by hypoxia, and can be used as a safe and reliable anti-hypoxia medicament, health-care product or food.)

1. Application of chai dalwood fatly mushroom phenolic acid in preparing medicine, health product or food for protecting acute and chronic anoxia injury.

2. Use according to claim 1, characterized in that: the medicine, health-care product or food for protecting acute and chronic hypoxia injury is a medicine, health-care product or food for improving the hypoxia tolerance of plateau patients and the acute hypoxia injury compensation capability of organisms of acute hypoxia people.

3. Use according to claim 1 or 2, characterized in that: the improvement of the hypoxia tolerance of the plateau patients and the acute hypoxia injury compensation capability of the organisms of the acute hypoxia people refers to the reduction of LDH and NO levels of the hypoxia people and the increase of ATP levels.

4. Use according to claim 2, characterized in that: the altitude diseases comprise chronic altitude diseases and acute altitude diseases.

5. Use according to claim 2, characterized in that: the patient suffering from the high altitude disease is a patient with impaired physical function when suffering from acute hypoxia and hypoxia tolerance.

6. Use according to any one of claims 1 to 5, characterized in that: the medicine, health-care product or food for protecting acute and chronic hypoxia injury is in an oral dosage form; further, the oral dosage form is selected from tablets, powders, capsules, pills, granules, powders, extracts or oral administration.

7. Use according to claim 1, characterized in that: the purity of the phenolic acid is 60-95% w/w, further 80-95% w/w, and further 90-93% w/w.

8. Use according to any one of claims 1 to 7, characterized in that: the chaihu fatlute phenolic acid is prepared by adopting an ultrasonic-assisted ethanol extraction method; further, the ultrasonic-assisted ethanol extraction method specifically operates as follows: pulverizing, sieving, removing sugar, extracting with ethanol under the assistance of ultrasonic wave to obtain phenolic acid crude extract, purifying with macroporous resin, collecting eluate, concentrating, and drying to obtain phenolic acid pure product; furthermore, the concentration of the extraction solvent ethanol is 85-95% v/v, the power of ultrasonic-assisted extraction is 160-200W, and the extraction temperature is 56-66 ℃; furthermore, the concentration of the extraction solvent ethanol is preferably 90 + -2% v/v, the power of ultrasonic-assisted extraction is preferably 180 + -5W, and the extraction temperature is preferably 61 + -2 ℃.

9. Use according to claim 8, characterized in that: the sugar removal operation is as follows: pulverizing the chaenomeles speciosa, sieving, degreasing, extracting with water and precipitating with ethanol, filtering the extracted residue, and volatilizing the solvent to obtain sugar-removed chaenomeles speciosa residue; the specific operation of the water extraction and alcohol precipitation method for removing sugar is as follows: dissolving the sieved powder of the large-fat faggot mushrooms in water, filtering to obtain filter residues, namely the sugar-removed residues of the large-fat faggot mushrooms, adding ethanol into the filtrate during filtering until the ethanol content reaches 70-90% v/v, standing, and filtering completely after precipitation; further, sugar is removed, and ethanol is added until the alcohol content is 75-85% v/v, preferably 80 +/-2% v/v.

10. Use according to claim 8, characterized in that: the specific operation of purifying the crude phenolic acid extract by macroporous resin is as follows: pretreating D101 macroporous adsorption resin, wherein the sampling concentration of crude phenolic acid is 10-30 mg/mL, the sampling volume is 0.2-0.4 BV, and the elution speed is 0.2-0.4 mL/min; further, it is preferable that the loading concentration is 20. + -.2 mg/mL, the loading volume is 0.3. + -. 0.05BV, and the elution rate is 0.3. + -. 0.05 mL/min.

11. Use according to any one of claims 1 to 10, characterized in that: the chai dalwood fat mushroom is a chai dalwood double-layer ring mushroom fruiting body.

Technical Field

The invention relates to the technical field of medicinal health products, in particular to an anti-hypoxia application of faggot fatly mushroom phenolic acid.

Technical Field

High Altitude Disease (HAD) is an idiopathic disease occurring in a High altitude hypoxic environment (generally, altitude above 3000 m), and High altitude hypoxic is a major pathogenic factor. The altitude diseases can be divided into two major groups, namely acute altitude disease (AMS) and Chronic altitude disease (CMS), according to the urgency of onset: AMS refers to various clinical syndromes occurring within hours to days when entering a plateau from a plain or entering a higher-altitude area from a plateau, including acute mild altitude disease, high altitude pulmonary edema, and high altitude cerebral edema; CMS refers to the clinical syndrome of life or immigration living in plateau for a long time, which is caused by the gradual loss of habituation to plateau hypoxic environment, and comprises 4 subtypes, such as plateau erythrocytosis, plateau heart disease, mixed chronic plateau disease, plateau recession and the like. Under the condition of acute hypoxia, due to the stimulation of hypoxia signals, a series of compensatory reactions such as accelerated respiration, vasoconstriction, heart rate increase and the like can occur in order to adapt to the hypoxic environment, so that the oxygen supply of important organs such as brain, heart and the like is ensured. Under the condition of oxygen deficiency, the preparation can promote the generation of red blood cells and blood vessels of the body, enhance glycolysis and glucose transport and enable the body to generate hypoxia tolerance. Promoting the adaptability of the organism under the condition of oxygen deficiency is an important measure for improving the hypoxia tolerance of the human body. The effective means is to perform hypoxia pre-adaptation and ingest hypoxia-resistant medicines or functional foods, the hypoxia pre-adaptation needs to be supported by specific equipment and takes a long time, and the anti-hypoxia medicines or foods have the characteristics of convenience in carrying, rapid action and the like. LDH is one of key enzymes in the anaerobic metabolism process, has important influence on energy metabolism of organisms, tissues and cells under the anaerobic condition, and the activity of the LDH is related to the generation of anaerobic metabolism energy of the organisms and the tolerance capability to oxygen deficiency; under continuous hypoxic conditions, ATP levels drop due to insufficient supply of substrate NO during mitochondrial oxidative phosphorylation, and brain tissue replenishes energy by increasing levels of anaerobic metabolism.

The chai fatigues sinensis is also called chai bi-layer Agaricus (Agaricus bitorquis (qui.) sacchaidamu.), mainly grows below 20-70 cm of a gobi soil layer with the elevation of 2600-3300 m in a Qinghai chai wood basin, is rich in various anti-hypoxia active ingredients such as phenolic acid and the like, and is widely eaten by local farmers and herders as natural anti-hypoxia food. At present, the anti-hypoxia report of the chaulmoogra fat mushroom phenolic acid is not found.

Disclosure of Invention

The invention aims to provide a new application of chaulmoogra fat mushroom phenolic acid.

Specifically, the invention provides application of chaulmoogra fat mushroom phenolic acid in preparation of medicines, health-care products or foods for protecting acute and chronic anoxic injuries.

Furthermore, the medicine, health-care product or food for protecting acute and chronic hypoxia injury is a medicine, health-care product or food for improving the hypoxia tolerance of plateau patients and the acute hypoxia injury compensation capability of organisms of acute hypoxia people.

Wherein, the improvement of the hypoxia tolerance of the plateau patients and the acute hypoxia injury compensation capability of the organisms of the acute hypoxia people refers to the reduction of LDH and NO levels of the hypoxia people and the increase of ATP levels.

Wherein the altitude disease comprises chronic altitude disease and acute altitude disease.

Further, the patients suffering from high altitude disease are patients with impaired body functions when suffering from acute hypoxia and hypoxia tolerance.

Wherein, the medicine, health care product or food for protecting acute and chronic hypoxia injury is an oral preparation; further, the oral dosage form is selected from tablets, powders, capsules, pills, granules, powders, extracts or oral administration.

The purity of the phenolic acid is 60-95% w/w, further 80-95% w/w, and further 90-93% w/w.

The chaihu fatlute phenolic acid is prepared by adopting an ultrasonic-assisted ethanol extraction method; further, the ultrasonic-assisted ethanol extraction method specifically operates as follows: pulverizing, sieving, removing sugar, extracting with ethanol under the assistance of ultrasonic wave to obtain phenolic acid crude extract, purifying with macroporous resin, collecting eluate, concentrating, and drying to obtain phenolic acid pure product; furthermore, the concentration of the extraction solvent ethanol is 85-95% v/v, the power of ultrasonic-assisted extraction is 160-200W, and the extraction temperature is 56-66 ℃; furthermore, the concentration of the extraction solvent ethanol is preferably 90 + -2% v/v, the power of ultrasonic-assisted extraction is preferably 180 + -5W, and the extraction temperature is preferably 61 + -2 ℃.

The sugar removal operation is as follows: pulverizing the chaenomeles speciosa, sieving, degreasing, extracting with water and precipitating with ethanol, filtering the extracted residue, and volatilizing the solvent to obtain sugar-removed chaenomeles speciosa residue; the specific operation of the water extraction and alcohol precipitation method for removing sugar is as follows: dissolving the sieved powder of the large-fat faggot mushrooms in water, filtering to obtain filter residues, namely the sugar-removed residues of the large-fat faggot mushrooms, adding ethanol into the filtrate during filtering until the ethanol content reaches 70-90% v/v, standing, and filtering completely after precipitation; further, sugar is removed, and ethanol is added until the alcohol content is 75-85% v/v, preferably 80 +/-2% v/v.

The specific operation of purifying the crude phenolic acid extract by macroporous resin is as follows: pretreating D101 macroporous adsorption resin, wherein the sampling concentration of crude phenolic acid is 10-30 mg/mL, the sampling volume is 0.2-0.4 BV, and the elution speed is 0.2-0.4 mL/min; further, it is preferable that the loading concentration is 20. + -.2 mg/mL, the loading volume is 0.3. + -. 0.05BV, and the elution rate is 0.3. + -. 0.05 mL/min.

The chaenomeles speciosa phenolic acid is an alcohol extract of a fruiting body of double-layer Agaricus bisporus (Quel.) Sacc Chaidatamu, and phenolic acid is taken as a main component in the alcohol extract. The determination in the specific embodiment of the invention shows that the phenolic acid content in the alcohol extract is 91 +/-2% w/w, the purity of the phenolic acid is higher, the biological activity experiment of the phenolic acid with the purity can be carried out, the result can truly reflect the biological activity of the chaulmoogra fat mushroom phenolic acid, and the interference of other components on the determination of the phenolic acid activity is effectively avoided. On the basis of the known biological activity of the phenolic acid, even if the content of the phenolic acid in the extract is reduced, the same or similar biological activity can be exerted as long as the dosage of the extract is properly adjusted to enable the phenolic acid to reach the corresponding effective concentration. Therefore, the content of phenolic acid in the extract is limited to 60-95% w/w.

The research of the invention shows that the chaulmoogra fatliquor phenolic acid has the function of prolonging the death time of an anoxic mouse in a normal-pressure hypoxia-resistant test; prolonging the survival time of poisoned mice in a sodium nitrite poisoning test; the function of prolonging the wheezing time of an acute hypoxia mouse in an acute cerebral ischemic hypoxia test; can reduce LDH (lactate dehydrogenase) content and NO (carbon monoxide) content under the anoxic condition, increase ATP (adenosine triphosphate) content, enhance the tolerance capacity of hypoxia injury of organisms, improve the compensation capacity of acute hypoxia injury of tissues, protect the tissues from being damaged by hypoxia, and can be used as a safe and reliable anti-hypoxia medicament, health-care product or food.

Drawings

FIG. 1 is a statistical graph of LDH level, which is a result of a mouse hypoxia tolerance test at normal pressure;

FIG. 2 is a statistical chart of ATP levels, which is the result of the mouse hypoxia tolerance test at normal pressure;

FIG. 3 is a statistical graph of mouse normbaric hypoxia tolerance test results-NO levels.

Detailed Description

The invention is further illustrated by the following specific embodiments in conjunction with the accompanying drawings:

the chaihood fatigued mushroom phenolic acid used in the specific embodiment of the invention is prepared by adopting a conventional phenolic acid extraction and purification mode, and the specific operation in the invention is as follows:

preparation of chai dalwood fat mushroom phenolic acid

1) Sugar removal of large-fat mushrooms of chai da wood: respectively mixing the fruiting body powder with distilled water at a ratio of 1:20, maintaining at 70 deg.C for 60min, performing ultrasonic extraction, vacuum filtering, centrifuging to obtain residue, filtering the extractive solution, and discarding;

2) extracting the chai dalwood fat mushroom phenolic acid: weighing the residue of the large-fat mushroom without the sugar obtained in the step 1), mixing the residue with 90% ethanol, performing ultrasonic-assisted extraction, centrifuging to obtain an extracting solution, and filtering the residue and discarding; concentrating the extracting solution to obtain a crude phenolic acid extracting solution.

Second, purification of chai da mu fat mushroom phenolic acid

1) And (3) decoloring the crude phenolic acid extracting solution: and (3) sequentially decolorizing the crude phenolic acid extract by using activated carbon and removing proteins by using a Savege method, and purifying by using a macroporous adsorption resin-D101 chromatographic column and 90% ethanol as an eluent to obtain a purified phenolic acid ethanol extract.

The chaihu fatlute phenolic acid prepared by the method is measured by taking gallic acid as a standard substance, wherein the content of the phenolic acid is 90% +/-2 w/w.

Experiment of protection of Lidamia macrophylla phenolic acid against acute and chronic hypoxia injury

1) The evaluation test basis of the hypoxia resistance is as follows: according to the 'evaluation program and detection method of health food functionality' published by the ministry of health in China in 2003, 10 males and females (raised in cages) in each group are randomly divided into 1 blank control group, 1 positive (salidroside) control group and 3 dosage groups of 1 drug (total phenolic acid) according to the weight, and 7 groups are counted. One of the medium doses (150mg/kg) was 10 times the daily recommended intake per kg body weight, and the other 2 doses were made as high dose (300mg/kg) and low dose (75mg/kg) as follows, depending on the particular condition of the test substance. And detecting the contents of ATP, NO and LDH in the brain tissue according to the kit instruction.

2) Mouse normal pressure hypoxia tolerance test: continuously administering for 20-30 days through mouth, placing each group of mice into 250mL of wide-mouth bottle containing 15g of steel lime (1 mouse is placed in each bottle) 1h after the test object is administered last time, smearing the bottle mouth with vaseline, tightly covering to make it leak-proof, immediately timing, and observing the death time of the mice due to anoxia by using the stopping respiration as an index.

3) Sodium nitrite poisoning test: preparing 20mL of 200% sodium nitrite solution with the concentration of 2000mg/mL, performing intragastric lavage on the mice according to the weight of each mouse and 200-240 mg/kg BW of the mice, immediately timing, and recording the survival time of the mice.

4) Acute cerebral ischemia test: the mice are killed by cutting off the heads quickly, the mouth-opening wheezing condition of the mice is observed, the mouth-opening wheezing is not stopped within 5s as an observation index, and the wheezing time of the mice is recorded.

5) Evaluation test result of anti-hypoxia activity of chaenomeles speciosa phenolic acid

a mouse hypoxia tolerance test under normal pressure

TABLE 1 mouse results of the normbaric hypoxia tolerance test-death time

(n＝20)

As can be seen from Table 1: the low, medium and high dose groups of the phenolic acid test group can obviously prolong the death time of the hypoxia mice, compared with a blank control group and a positive control group (salidroside group), the death time difference of the normal pressure hypoxia-resistant test mice is obvious (P <0.05), and the following can be judged: the normal pressure hypoxia tolerance test result of the chai dalwood fatlute phenolic acid is positive.

b sodium nitrite poisoning test

Table 2 sodium nitrite poisoning test results-survival time

(n＝20)

Note: lower case letters indicate p <0.05 significance, the following.

Table 2 shows that: in the sodium nitrite poisoning test, the phenolic acid test group can prolong the survival time of the poisoning mice, the difference is significant (P is less than 0.05) compared with a blank and positive (salidroside) control group, and the low, medium and high dose groups of the phenolic acid test group have higher efficiency of prolonging the survival time of the poisoning mice compared with the blank and positive control group, and can be judged: the result of the sodium nitrite poisoning test of the chai dalwood fatlute phenolic acid is positive.

c acute cerebral ischemic hypoxia assay

TABLE 3 acute cerebral ischemic hypoxia test results-wheezing time(n＝20)

As can be seen from Table 3: in the acute cerebral ischemic hypoxia test, compared with a blank control group and a positive control group, each test group can obviously prolong the wheezing time difference of acute hypoxic mice (P <0.05), and compared with the blank control group and the positive control group, the total phenolic acid test group can also obviously prolong the wheezing time difference (P < 0.05). It can be judged that: the result of the acute cerebral ischemic hypoxia test of the chaenomeles speciosa phenolic acid is positive.

The dosage groups of the chai dalwood fatly alutamide phenolic acid have the effects of regulating and prolonging the survival time of mice in a normal pressure hypoxia test, a sodium nitrite poisoning test and an acute cerebral ischemic hypoxia test, are in dosage effect relationship, and have significant difference (P <0.05) between a high dosage group, a medium dosage group and a low dosage group and a blank model group, namely the results of the three determined anti-hypoxia tests are positive, so that the chai dalwood fatly alutamide phenolic acid is determined to have anti-hypoxia activity. And the anti-hypoxia effect of the phenolic acid high-dose group is obviously higher than that of the positive control group (rhodiola rosea dry), so the faggaretia phenolic acid can replace salidroside, and the problem of resource shortage in developing anti-hypoxia drugs is solved.

6) The influence of chaenomeles speciosa phenolic acid on relevant biochemical indicators is shown in fig. 1-3.

The chai dalwood fatlute phenolic acid dosage groups have the effects of regulating and reducing LDH and NO levels in a normal pressure hypoxia test, a sodium nitrite poisoning test and an acute cerebral ischemic hypoxia test, have the effects of regulating and increasing ATP levels, are in a dosage effect relationship, and have significant difference (p is less than 0.05) between a high dosage group and a model group.

The present invention has been described in detail, and it should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.