Brassica rapa alkali extract with anti-hypoxia activity as well as preparation method and application of brassica rapa alkali extract

Abstract

The invention discloses a brassica rapa alkali extract with anti-hypoxia activity as well as a preparation method and application of the brassica rapa alkali extract. The method comprises the following steps: by taking brassica rapa tuberous roots as a raw material, rinsing, slicing, drying, crushing, extracting with a food-grade calcium oxide alkaline solution, carrying out centrifugal separation to obtain supernate, and carrying out spray drying on the supernate to obtain the brassica rapa alkali extract. The method is easy to operate, low in cost and high in extraction rate, starch and crude fiber auxiliary components in the brassica rapa tubers can be removed to a large extent, the most important components with health-care functions of brassica rapa are reserved, and the biological activity of nutritional components of brassica rapa is kept. Results of evaluation and biochemical index determination of a normal-pressure closed experiment and a sodium nitrite poisoning experiment show that the brassica rapa alkali extract can remarkably prolong the survival time of mice, improve the activity of superoxide dismutase in the mice and reduce the content of malondialdehyde, and shows a relatively good hypoxia tolerance effect. The alkali extract can be used for developing hypoxia-resistant functional foods such as tabletted candies and biscuits, and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the preparation of a chamacarpine extract with hypoxia-resistant activity and its application in hypoxia-resistant food, belonging to the field of special food research. Background Art

[0002] Hypoxia is a pathological change process in which the body's function, metabolism and morphological structure undergo abnormal changes due to reduced oxygen supply or inability of tissues to utilize oxygen, including circulatory, hematologic, hypotonic and tissue hypoxia. The effects of hypoxia on various organs of the human body are related to the duration of hypoxia, the state of the body and the severity of hypoxia. The main symptoms of hypoxia include dizziness, headache, nausea, behavioral disorders, poor sleep quality, dyspnea, memory deterioration, etc. Being in a hypoxic environment for a long time can cause damage to important organs such as the lungs, brain, heart, and gastrointestinal tract, and in severe cases can lead to death. Therefore, studying the occurrence of hypoxia and the physiological changes caused by hypoxia is of great significance for the prevention of hypoxia (Li XF, Zhang JH, Li GA, et al. Highaltitude hypoxia and oxidative stress: The new hope brought by free radicalscavengers [J]. Life sciences, 2023, 336: 122319-122319).

[0003] Common hypoxia-tolerant chemical drugs, such as acetazolamide and dexamethasone, have many adverse reactions and allergic reactions, and long-term use is harmful to health. Therefore, researchers have shifted their research focus to drugs of natural origin. Studies have found that a variety of Tibetan medicines have certain therapeutic effects on body damage caused by plateau hypoxia, such as Pteridium anserine, Ganoderma lucidum, Gastrodia elata, Acorus calamus, Cordyceps sinensis, Rhodiola rosea, etc. (Wang Zihan, Zhao Anpeng, Mou Hongfang, et al. Research Progress on the Protective Effect of Tibetan Medicine on Plateau Hypoxia Damage [J]. Pharmaceutical Practice and Service, 2022, 40(05): 408-415) At present, the research on Tibetan medicine in hypoxia tolerance is basically in the preliminary pharmacodynamics, and there is a problem of insufficient in-depth research, and the research on the active ingredients and mechanism of action of the drugs is still unclear. In addition, most of the well-known hypoxia Tibetan medicines are facing the problems of reduced natural resources and immature cultivation technology. Therefore, exploring and developing safe hypoxia-tolerant drugs from Tibetan medicine is a promising research strategy.

[0004] Chamagu (Brassica rapa.L) is called "Niuma" in Tibetan, also known as turnip and pancai. It belongs to the Brassica rapa subspecies and is a plateau plant that grows above 3,200 meters above sea level. Chamagu likes cool weather, tolerates light frost, and is not resistant to heat. It is a long-day plant. Chamagu is mainly produced in the southwest of Tianshan Mountain in Xinjiang and the northwest of Tarim. It is rich in calcium, iron, zinc, potassium and other mineral elements (Zhang Shiwen. ICP-OES determination of calcium, iron and phosphorus in Chamagu [J]. Xinjiang Nonferrous Metals, 2019, 42(04): 15-17), as well as raw polysaccharides, flavonoids, alkaloids, and glucosinolate compounds (Zhang Tao, An Xiqiang, Cheng Jiangnan, et al. Research progress on the chemical composition and biological activity of the Uyghur medicine Chamagu [J]. Chinese Pharmacy, 2017, 28(07): 982-986). Studies have also found that Chamagu has anti-inflammatory, liver protection, cough and phlegm relief, dampness and detoxification, anti-radiation, and hypoxia resistance (Wang Yu, Wang Zhang, Kuang Tingting, et al. Research on the anti-plateau hypoxia effect of turnip polysaccharide [J]. Journal of Chengdu University (Natural Science Edition), 2014, 33(02): 115-117).

[0005] As a special medicinal and edible plant in Xinjiang, my country, Chamagu has richer active substances and good development prospects. Wang Yu et al. gavaged mice with Chamagu polysaccharide, and through normal pressure closed hypoxia experiment and low pressure closed hypoxia experiment and determination of MDA content and SOD activity in mouse brain tissue and serum, proved that Chamagu polysaccharide has the effect of improving the body's ability to scavenge free radicals and improve hypoxia tolerance (Wang Yu, Wang Zhang, Kuang Tingting, et al. Study on the anti-plateau hypoxia effect of rapa polysaccharide [J]. Journal of Chengdu University (Natural Science Edition), 2014, 33(02): 115-117).

[0006] Kuang Nanyue conducted a mouse experiment with a high-dose group (0.7570 mg / mL) and a low-dose group (0.3785 mg / mL) of Chamagu flavonoids extract. The experimental results showed that the SOD activity in the brain of mice in both the high-dose and low-dose groups increased, the MDA content decreased significantly, and the GSH-Px in the high-dose group increased significantly, proving that the Chamagu brass extract has a significant antioxidant effect (Kuang Nanyue. Preliminary study on the quality standard of turnip medicinal materials [D]. Urumqi: Master's thesis of Xinjiang Medical University, 2011: 36). However, these studies cannot fully enrich the functional components of Chamagu and fully exert its hypoxia resistance function.

[0007] CN104095898A discloses a method for preparing Chamagu paste and its use in drugs for hypoxia at normal pressure (Chengdu University of Traditional Chinese Medicine. Use of a kind of turnip paste [P]. China: 201410349006.X.2014-10-15). It is to soak Chamagu slices, decoct, filter, and then decoct the filter residue, repeat two to three times, collect the filtrate, and concentrate it to a relative density of 1.20 to 1.45 to prepare an extract. In the animal hypoxia tolerance test, the survival time of mice in the Chamagu paste group was longer than that in the blank group. The feeding dose of the patent sample is high, and the hypoxia tolerance effect is average.

[0008] Yang Yongdong used the best extraction method with a solid-liquid ratio of 1:30g / mL, 90℃ water bath reflux extraction 3 times, 2h each time, and 30% activated carbon decolorization to obtain Chamagu polysaccharide, and conducted animal experiments to test the protective effect of Chamagu polysaccharide on normal pressure hypoxia and acute hypoxia-hypoxia injury in mice. The results of the normal pressure hypoxia experiment in mice showed that the high-dose group of Chamagu polysaccharide could prolong the hypoxia tolerance time, increase the SOD activity in brain tissue and serum, and reduce the MDA content. The results showed that Chamagu polysaccharide has the effect of hypoxia tolerance and protection against hypoxia injury (Yang Yongdong. Preparation, component analysis and anti-acute hypoxia-hypoxia injury effect of Tibetan medicine turnip polysaccharide [D]. Chengdu: Chengdu University of Traditional Chinese Medicine, 2014).

[0009] Zhang Liming et al. extracted yam glycoprotein using a slightly alkaline solution, and the highest extraction rate was 0.74% when the extraction conditions were a temperature of 60°C, an extraction time of 2.0 h, a solid-liquid ratio of 1:15 g / mL, and a pH value of 10. A slightly alkaline environment can extract glycoproteins from yam as much as possible, while a strong alkaline environment will cause a greater degree of hydrolysis and destruction of glycoproteins, resulting in a decrease in the extraction rate (Zhang Liming, Wang Yanqiao, Zuo Beimei, et al. Study on the process of extracting yam glycoprotein by a slightly alkaline method [J]. Food Science and Technology, 2012, 37(03): 206-209).

[0010] CN114053390A discloses a hypoxia-resistant Chinese medicine composition and its preparation method and application, wherein the Chinese medicine composition includes 20-55 parts of Rhodiola rosea, 18-25 parts of Potentilla anserina, 10-20 parts of Turnip, 5-10 parts of Corydalis yanhusuo, 5-10 parts of ginger, 5-10 parts of Lycium barbarum, and 5-10 parts of Astragalus membranaceus (Tibet University of Tibetan Medicine, Jiangxi University of Traditional Chinese Medicine. A hypoxia-resistant Chinese medicine composition and its preparation method and application [P]. China: 202111558037.2, 2022-02-18) The results of the normal pressure hypoxia tolerance experiment and the sodium nitrite poisoning experiment show that Turnip and Corydalis yanhusuo are added to the composition, which can prolong the hypoxia tolerance time of mice and the tolerance time of acute cerebral ischemic hypoxia in mice. This patent uses a combination of Chinese medicines, and is extracted by decoction, and the relative content of functional ingredients is low.

[0011] The above patents disclose the application of several active ingredients of Chamagu in hypoxia resistance, but the research results have shortcomings and the experimental effects are average. Chamagu is a traditional Tibetan medicinal material and a characteristic plateau medicinal and edible plant. Studies have found that the polysaccharide components in Chamagu have hypoxia resistance. Therefore, Chamagu has good development and utilization value and can be used to treat plateau hypoxia-related diseases and develop functional foods with the effect of protecting hypoxia damage. This has important practical significance for the comprehensive and rational use of resources, improving the economic value of Chamagu, and promoting economic development in plateau areas. Through searching, no patent disclosure documents related to the patent application of the present invention have been found. Summary of the invention

[0012] The purpose of the present invention is to extract the active ingredients of Chamagu using an alkali method and provide its application in hypoxia tolerance activity. The specific preparation steps of the Chamagu alkali extract of the present invention are as follows:

[0013] (1) Wash the raw material of Chamagugu with clean water, drain the water, slice it, place it in a vacuum drying oven at 50° C., grind it, and pass it through a 60-mesh sieve to obtain Chamagugu powder;

[0014] (2) adding the chamagu powder obtained in step (1) into an extraction tank, adding purified water in a mass ratio of chamagu powder to purified water of 1:15 to 1:25 g / mL, adjusting the pH of the extract to 7.0 to 8.0 with a food-grade calcium oxide mixed solution, setting the extraction temperature to 30° C. to 50° C., and extracting for 1 to 2 hours under this condition;

[0015] (3) After extraction, use a centrifuge to separate the solid and liquid, set the centrifuge speed to 8000 r / min, centrifuge for 15 min, and collect the supernatant;

[0016] (4) spray drying the supernatant obtained in step (3) at an inlet temperature of 120° C., an outlet temperature of 56° C. to 60° C., a fan speed of 97%, a peristaltic pump speed of 30%, and a needle-passing interval of 5 s to obtain an anoxic-resistant chamaque alkaloid extract.

[0017] The beneficial effects achieved by the present invention are:

[0018] (1) The present invention can remove the secondary chemical components of chamaguchi starch and crude fiber to the maximum extent, and retain the most important nutrients with health functions, including soluble chamaguchi protein, chamaguchi polysaccharides, chamaguchi flavonoids, chamaguchi polyphenols, vitamins and various mineral nutrients.

[0019] (2) The main active ingredients in the chaga alkaloid extract extracted by the present invention can be greatly concentrated and enriched, and the solubility performance is also significantly improved. The obtained chaga alkaloid extract significantly improves the survival time of mice in the normal pressure closed hypoxia experiment and the sodium nitrite poisoning experiment compared with the blank group; at the same time, it can reduce the content of malondialdehyde (MDA) in the heart, brain and lung tissues, and increase the content of superoxide dismutase (SOD).

[0020] (3) The traditional alkaline extraction method has too high extraction temperature or pH value, which not only easily denatures the protein and affects the functional properties of the product, but also increases the viscosity of the crude protein mother liquor, making separation difficult and increasing losses. When processed at high temperatures, the protein will undergo chemical changes such as racemization, hydrolysis, desulfurization and deamidation, most of which are irreversible. In contrast, the present invention has the advantages of short extraction time, mild conditions, low energy consumption, and suitability for large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Raw materials for preparing hypoxia-tolerant Chamagu alkaline extract (A Chamagu plant B Chamagu root tuber)

[0022] Figure 2 Process flow chart for preparing hypoxia-resistant Chamagold alkali extract

[0023] Figure 3 Chamagu powder and hypoxia-tolerant Chamagu alkali extract (A Chamagu powder B Chamagu alkali extract)

[0024] Figure 4 Hematoxylin-eosin staining of lung tissues of mice in a normal pressure closed experiment after being fed with low-dose chamacarpine extract (A1 blank control group, A2 partial enlargement of blank control group; B1 salidroside group, B2 partial enlargement of salidroside group; C1 low-dose chamacarpine extract group, C2 partial enlargement of low-dose chamacarpine extract group)

[0025] Figure 5 Hematoxylin-eosin staining of lung tissues of mice in a normal pressure closed experiment after feeding with a medium dose of chamacha alkaloid extract (A1 blank control group, A2 partial enlargement of the blank control group; B1 salidroside group, B2 partial enlargement of the salidroside group; D1 medium dose group of chamacha alkaloid extract, D2 partial enlargement of the medium dose group of chamacha alkaloid extract)

[0026] Figure 6 Hematoxylin-eosin staining of lung tissues of mice in a normal pressure closed experiment after being fed with a high dose of chamacha alkaloid extract (A1 blank control group, A2 partial enlargement of the blank control group; B1 salidroside group, B2 partial enlargement of the salidroside group; E1 high-dose chamacha alkaloid extract group, E2 partial enlargement of the high-dose chamacha alkaloid extract group) DETAILED DESCRIPTION

[0027] In order to better understand the present invention, the present invention is further described in detail below in conjunction with embodiments, but the scope of protection claimed by the present invention is not limited to the scope represented by the embodiments.

[0028] The following experimental procedures were used throughout the examples:

[0029] (1) Glucose was used as the standard reference substance and the phenol-sulfuric acid method was used to determine the polysaccharide content of the chamaeko alkaloid extract.

[0030] (2) The protein content of chamaemorin extract was determined by the G-250 Coomassie Brilliant Blue method using bovine serum albumin as the standard reference substance.

[0031] (3) Gallic acid was used as the standard reference substance and the Folin-phenol method was used to determine the total polyphenol content of the chamaeko alkaloid extract.

[0032] (4) Using rutin as the standard reference substance, the sodium nitrite-aluminum nitrate method was used to determine the total flavonoids content of the chamaecyparis extract.

[0033] The present invention will be further described below with reference to examples.

[0034] Embodiment 1:

[0035] The specific preparation steps of the chamacha alkali extract used in this embodiment are as follows:

[0036] The root tubers of Chamagu were washed with clean water, sliced ​​after draining, placed in a vacuum drying oven at 50°C for drying, crushed, and passed through a 60-mesh sieve to obtain Chamagu powder. Weigh 50g of Chamagu powder and add 750g of pure water to the extraction tank, adjust the pH of the extract to 6.5 with a food-grade calcium oxide mixture, set the extraction temperature to 30°C, and extract for 1.0h under this condition. After the extraction is completed, set the centrifuge speed to 8000r / min for 15min, collect the supernatant, dilute and spray dry, set the inlet temperature to 120°C, the outlet temperature to 56°C~60°C, the fan speed to 97%, the peristaltic pump speed to 30%, and the needle-passing interval to 5s. Under this condition, the Chamagu alkali extract was obtained. The polysaccharide content in the Chamagu alkali extract was 50.1%, the protein content was 0.1%, the polyphenol content was 0.32%, and the flavonoid content was 0.05%.

[0037] Embodiment 2:

[0038] The specific preparation steps of the chamacha alkali extract used in this embodiment are as follows:

[0039] The root tubers of Chamagu were washed with clean water, sliced ​​after draining, placed in a vacuum drying oven at 50°C for drying, crushed, and passed through a 60-mesh sieve to obtain Chamagu powder. Weigh 50g of Chamagu powder and add 750g of pure water to the extraction tank, adjust the pH of the extract to 7.0 with a food-grade calcium oxide mixture, set the extraction temperature to 30°C, and extract for 1.0h under this condition. After the extraction is completed, set the centrifuge speed to 8000r / min for 15min, collect the supernatant, dilute and spray dry, set the inlet temperature to 120°C, the outlet temperature to 56°C~60°C, the fan speed to 97%, the peristaltic pump speed to 30%, and the needle-passing interval to 5s. Under this condition, the Chamagu alkali extract was obtained. The polysaccharide content in the Chamagu alkali extract was 52.1%, the protein content was 0.3%, the polyphenol content was 0.35%, and the flavonoid content was 0.05%.

[0040] Example 3

[0041] The specific preparation steps of the chamacha alkali extract used in this embodiment are as follows:

[0042] The root tubers of Chamagu were washed with clean water, sliced ​​after draining, placed in a vacuum drying oven at 50°C for drying, crushed, and passed through a 60-mesh sieve to obtain Chamagu powder. Weigh 50g of Chamagu powder and add 750g of pure water to the extraction tank, adjust the pH of the extract to 7.5 with a food-grade calcium oxide mixture, set the extraction temperature to 30°C, and extract for 1.0h under this condition. After the extraction is completed, set the centrifuge speed to 8000r / min for 15min, collect the supernatant, dilute and spray dry, set the inlet temperature to 120°C, the outlet temperature to 56°C~60°C, the fan speed to 97%, the peristaltic pump speed to 30%, and the needle-passing interval to 5s. Under this condition, the Chamagu alkali extract was obtained. The polysaccharide content in the Chamagu alkali extract was measured to be 53.4%, the protein content was 0.34%, the polyphenol content was 0.37%, and the flavonoid content was 0.06%.

[0043] Example 4

[0044] The specific preparation steps of the chamacha alkali extract used in this embodiment are as follows:

[0045] The root tubers of Chamagu were washed with clean water, sliced ​​after draining, placed in a vacuum drying oven at 50°C for drying, crushed, and passed through a 60-mesh sieve to obtain Chamagu powder. Weigh 50g of Chamagu powder and add 750g of pure water to the extraction tank, adjust the pH of the extract to 8.0 with a food-grade calcium oxide mixture, set the extraction temperature to 30°C, and extract for 1.0h under this condition. After the extraction is completed, set the centrifuge speed to 8000r / min for 15min, collect the supernatant, dilute and spray dry, set the inlet temperature to 120°C, the outlet temperature to 56°C~60°C, the fan speed to 97%, the peristaltic pump speed to 30%, and the needle-passing interval to 5s. Under this condition, the Chamagu alkali extract was obtained. The polysaccharide content in the Chamagu alkali extract was measured to be 54.6%, the protein content was 0.46%, the polyphenol content was 0.4%, and the flavonoid content was 0.11%.

[0046] Example 5

[0047] The specific preparation steps of the chamacha alkali extract used in this embodiment are as follows:

[0048] The root tubers of Chamagu were washed with clean water, sliced ​​after draining, placed in a vacuum drying oven at 50°C for drying, crushed, and passed through a 60-mesh sieve to obtain Chamagu powder. Weigh 50g of Chamagu powder and add 750g of pure water to the extraction tank, adjust the pH of the extract to 8.5 with a food-grade calcium oxide mixture, set the extraction temperature to 30°C, and extract for 1.0h under this condition. After the extraction is completed, set the centrifuge speed to 8000r / min for 15min, collect the supernatant, dilute and spray dry, set the inlet temperature to 120°C, the outlet temperature to 56°C~60°C, the fan speed to 97%, the peristaltic pump speed to 30%, and the needle-passing interval to 5s. Under this condition, the Chamagu alkali extract was obtained. The polysaccharide content in the Chamagu alkali extract was 55.2%, the protein content was 0.41%, the polyphenol content was 0.42%, and the flavonoid content was 0.13%.

[0049] Example 6

[0050] The specific preparation steps of the chamacha alkali extract used in this embodiment are as follows:

[0051] The root tubers of Chamagu were washed with clean water, sliced ​​after draining, placed in a vacuum drying oven at 50°C for drying, crushed, and passed through a 60-mesh sieve to obtain Chamagu powder. Weigh 50g of Chamagu powder and add 750g of pure water to the extraction tank, adjust the pH of the extract to 9.0 with a food-grade calcium oxide mixture, set the extraction temperature to 30°C, and extract for 1.0h under this condition. After the extraction is completed, set the centrifuge speed to 8000r / min for 15min, collect the supernatant, dilute and spray dry, set the inlet temperature to 120°C, the outlet temperature to 56°C~60°C, the fan speed to 97%, the peristaltic pump speed to 30%, and the needle-passing interval to 5s. Under this condition, the Chamagu alkali extract was obtained. The polysaccharide content in the Chamagu alkali extract was measured to be 555.1%, the protein content was 0.39%, the polyphenol content was 0.40%, and the flavonoid content was 0.11%.

[0052] Embodiment 7:

[0053] The specific preparation steps of the chamacha alkali extract used in this embodiment are as follows:

[0054] The root tubers of Chamagu were washed with clean water, sliced ​​after draining, placed in a vacuum drying oven at 50°C for drying, crushed, and passed through a 60-mesh sieve to obtain Chamagu powder. Weigh 50g of Chamagu powder and add 1000g of pure water to the extraction tank, adjust the pH of the extract to 8.0 with a food-grade calcium oxide mixture, set the extraction temperature to 40°C, and extract for 1.0h under this condition. After the extraction is completed, set the centrifuge speed to 8000r / min for 15min, collect the supernatant, dilute and spray dry, set the inlet temperature to 120°C, the outlet temperature to 56°C~60°C, the fan speed to 97%, the peristaltic pump speed to 30%, and the needle-passing interval to 5s. Under this condition, the Chamagu alkali extract was obtained. The polysaccharide content in the Chamagu alkali extract was 56.34%, the protein content was 0.5%, the polyphenol content was 0.51%, and the flavonoid content was 0.15%.

[0055] Embodiment 8:

[0056] The specific preparation steps of the chamacha alkali extract used in this embodiment are as follows:

[0057] The tuber of Cichorium glandulosum Boiss. et Huet was washed with clean water, sliced after draining the water, dried in a vacuum drying oven at 50 °C, pulverized, and passed through a 60-mesh sieve to obtain Cichorium glandulosum powder. 50 g of Cichorium glandulosum powder was weighed and added to 1000 g of pure water and fed into an extraction tank. The pH of the extract was adjusted to 8.0 with a food-grade calcium oxide mixture. The extraction temperature was set at 40 °C, and extraction was carried out under these conditions for 1.5 h. After extraction, the centrifuge was set at a speed of 8000 r / min and centrifuged for 15 min. The supernatant was collected, diluted and spray-dried. The inlet temperature was set at 120 °C, the outlet temperature was 56 °C - 60 °C, the fan speed was 97%, the peristaltic pump speed was 30%, and the needle-passing interval time was 5 s. Under these conditions, the alkali extract of Cichorium glandulosum was prepared. The polysaccharide content in the alkali extract of Cichorium glandulosum was measured to be 55.2%, the protein content was 0.5%, the polyphenol content was 0.42%, and the flavonoid content was 0.13%.

[0058] Example 9

[0059] The specific preparation steps of the alkali extract of Cichorium glandulosum used in this example are as follows:

[0060] The tuber of Cichorium glandulosum Boiss. et Huet was washed with clean water, sliced after draining the water, dried in a vacuum drying oven at 50 °C, pulverized, and passed through a 60-mesh sieve to obtain Cichorium glandulosum powder. 50 g of Cichorium glandulosum powder was weighed and added to 1200 g of pure water and fed into an extraction tank. The pH of the extract was adjusted to 8.0 with a food-grade calcium oxide mixture. The extraction temperature was set at 50 °C, and extraction was carried out under these conditions for 2.0 h. After extraction, the centrifuge was set at a speed of 8000 r / min and centrifuged for 15 min. The supernatant was collected, diluted and spray-dried. The inlet temperature was set at 120 °C, the outlet temperature was 56 °C - 60 °C, the fan speed was 97%, the peristaltic pump speed was 30%, and the needle-passing interval time was 5 s. Under these conditions, the alkali extract of Cichorium glandulosum was prepared. The polysaccharide content in the alkali extract of Cichorium glandulosum was measured to be 55.16%, the protein content was 0.49%, the polyphenol content was 0.43%, and the flavonoid content was 0.12%.

[0061] Example 10

[0062] Anti-hypoxia pharmacodynamic experiment

[0063] (1) Experimental grouping and administration

[0064] 60 SPF-grade inbred CD-1 male mice, weighing 20 ± 2 g, experimental animal license number: [SCXK(Beijing)2021-0006], were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. After 1 week of adaptive feeding, the mice were randomly divided into 5 groups (n = 12 / group): blank control group, salidroside group at 150 mg / kg, low-dose Cichorium glandulosum group at 300 mg / kg, medium-dose Cichorium glandulosum group at 600 mg / kg, high-dose Cichorium glandulosum group at 900 mg / kg;

[0065] The chaga alkaloid extract prepared in Example 7 was fully dispersed and dissolved in distilled water, and distilled water was used as a blank control group. After 28 days of oral administration, 6 mice were randomly selected from each group for a normal pressure hypoxia tolerance test, and the other 6 mice were tested for a sodium nitrite poisoning test. During the feeding and administration period, the mice were allowed to drink water and eat freely, and the weight of the mice was recorded.

[0066] The experimental results showed that after 28 days of administration, the weight of mice increased, and there was no significant difference in weight between the control group and the administration group. The organ coefficient is an important indicator of animal organ development, which can directly reflect the toxic effects of the test substance on animals. The results are shown in Table 1. There was no significant difference between the administration group and the control group (P>0.05). In summary, the extract of Chaga alkaloids does not cause changes in appetite in experimental mice, affect body weight and organ lesions.

[0067] Table 1 Body weight and organ index of experimental mice

[0068]

[0069] (2) Normal pressure sealing test

[0070] The evaluation was performed with reference to the method reported by Xu et al. (Xu L, Mi JM, Pu XM, et al. Protective effect and mechanism of Polygonatum kingianum against hypoxia-induced injury [J]. Heliyon, 2023, 9 (3): e14353). The specific method is to place the mouse in a 200 mL wide-mouth bottle, take 2 g of soda lime and put it in the jar to absorb carbon dioxide and moisture in the air. Finally, the bottle mouth is sealed with white vaseline, and the timing starts after sealing. Respiratory arrest is an indicator of mouse death. The time from the mouse being placed in the bottle to death is recorded. The results are shown in Table 2. The experimental results show that compared with the blank control group and the salidroside group, the survival time of mice in the high, medium and low dose groups of chamagu alkaloid extract was significantly increased, and the mice in the high dose group of chamagu alkaloid extract had the longest survival time.

[0071] Table 2 Effect of Chamagold alkaloid extract on the survival time of confined hypoxic mice

[0072]

[0073] (3) Sodium nitrite poisoning test

[0074] The evaluation was performed with reference to the method reported by Xie et al. (Xie Y, Jing SP, Su DH, et al. Composition analysis and anti-hypoxia activity of polysaccharide from Brassica rapa L [J]. International Journal of Biological Macromolecules, 2010, 47 (4): 528-533). The specific method is to intraperitoneally inject 5% sodium nitrite (200 mg / kg BW) into each mouse 1 hour after administration on the 28th day of feeding. The survival time of the mice was recorded, and the results are shown in Table 3. The experimental results showed that compared with the blank control group, the survival time of the high-dose administration group of Chamagu alkaloid extract was significantly increased. Compared with the salidroside group, the survival time of mice in the high-dose administration group of Chamagu was significantly increased.

[0075] Table 3 Effect of chamacha alkaloid extract on survival time of mice poisoned by sodium nitrite

[0076]

[0077] (4) Determination of oxidative stress indicators in mouse tissues

[0078] The separated heart, lung and liver tissues were added to the extract and placed in a tissue grinder for ice bath homogenization; after centrifugation at 8000r / min and 4℃ for 10min, the supernatant was taken and placed on ice for testing. According to the instructions of the malondialdehyde (MDA) and superoxide dismutase (SOD) kits, the relevant detection reagents were added, and the absorbance was measured by a microplate reader and the relevant content was calculated. The results of the effect of the Chamagu alkali extract on the MDA oxidative stress in the heart, lung and liver tissues of closed hypoxic mice are shown in Table 4. The results of the effect of the Chamagu alkali extract on the SOD oxidative stress in the heart, lung and liver tissues of closed hypoxic mice are shown in Table 5. It can be seen from the experimental results in Tables 4 and 5 that compared with the blank control group, the MDA content of the Chamagu experimental group decreased, and the SOD inhibition rate was significantly improved, indicating that the Chamagu alkali extract has the effect of improving the hypoxia tolerance of mice and protecting against oxidative stress damage.

[0079] Table 4 Effects of chaga extract on MDA oxidative stress in heart, lung and liver tissues of confined hypoxic mice

[0080]

[0081] Table 5 Effects of Chamagold extract on SOD oxidative stress in heart, lung and liver tissues of confined hypoxic mice

[0082]

[0083] (5) HE staining of mouse lung tissue

[0084] The lung tissue was fixed with 4% paraformaldehyde overnight, dehydrated with a series of graded alcohols, embedded in paraffin, sliced ​​(4 μm thick), dewaxed, dehydrated, and stained with hematoxylin and eosin. Lung tissue cells, alveolar wall capillaries, alveolar septa, alveolar structure, and inflammatory cell infiltration were observed under a microscope. The experimental results are shown in the attached Figures 4 to 6 . Figures 4 to 6 A1 in the middle is the blank control group. As can be seen from the local magnified area A2 of Figure A1, the lung tissue showed obvious alveolar wall thickening, neutrophil infiltration, and alveolar collapse. B1 is the salidroside group. As can be seen from the local magnified area B2 of Figure B1, the lung tissue showed mild alveolar wall thickening, inflammatory cell infiltration, and alveolar collapse. Figure 4 C1 is the low-dose group of chaga alkaloid extract. From the local enlarged area C2 of C1, it can be seen that the lung tissue showed neutrophil infiltration, alveolar collapse, thickening of alveolar septa, destruction and disorder of alveolar structure, and unclear layers. Figure 5 D1 is the medium-dose group of chamacarpine extract. From the local enlarged area D2 of D1, it can be seen that the lung tissue showed loose cell arrangement, obvious dilation and congestion of alveolar wall capillaries, and inflammatory cell infiltration. Figure 6 E1 is the high-dose group of chamacarpine extract. From the local enlarged area E2 of E1, it can be seen that the lung tissue showed loose cell arrangement and a small amount of inflammatory cell infiltration.

[0085] This study shows that chaga alkaloid extract can reduce the MDA content in the blood and increase the activity of SOD. On the one hand, it improves the body's ability to scavenge oxygen free radicals, and on the other hand, it reduces lipid peroxidation damage and plays a hypoxia-resistant role by reducing oxidative stress. HE staining of lung tissue showed that after feeding chaga alkaloid extract, the lung tissue cells of mice were filled, the swelling increased significantly, the boundary between the nucleus and the cytoplasm was clear, and the vacuoles around the nucleus were reduced.

[0086] Embodiment 11

[0087] 5 g of maltodextrin, 5 g of pregelatinized starch and 4 g of erythritol were added to the chamacha alkaloid extract, mixed evenly and then dried, 2 g of microcrystalline cellulose was added, mixed evenly and then sieved, 1 g of magnesium stearate and vitamin C were added and mixed evenly, the mixture was placed in a tablet press for pre-compression at a pressure of 15 kN, and tableted at a pressure of 60 kN to obtain chamacha alkaloid extract tablet candy.

[0088] Example 12

[0089] Active dry yeast and warm milk are mixed evenly in a mass ratio of 1:10 for later use. 500g of low-gluten flour, 100g of dregs powder, 20g of chamagu alkali extract, 1g of baking soda and 1.2g of salt are weighed, and the above raw materials are mixed with the yeast milk mixture and 30g of edible vegetable oil, and kneaded into dough. After the dough is proofed, it is made into a cake blank. The upper fire temperature is 200-250℃, the lower fire temperature is 250-300℃, and the baking time is 5min. The upper and lower fire temperatures are 180-200℃, and the baking time is 1min. The biscuits are made when the surface is golden.

[0090] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A method for preparing a chamacha alkaloid extract having hypoxia-resistant activity, characterized in that Follow the steps below to produce: (1) Wash the root tubers of Chamagugu with clean water, drain the surface water and slice them, dry them in an oven at 50° C., grind them and pass them through a 60-mesh sieve to obtain Chamagugu powder; (2) Adding chamagu powder to an extraction tank, adding purified water according to a mass ratio of chamagu powder to purified water of 1:15 to 1:25 g / mL, adjusting the pH of the extract to 7.0 to 8.0 with a food-grade calcium oxide mixture, setting the extraction temperature to 30° C. to 50° C., and extracting for 1 to 2 hours under this condition; (3) After extraction, use a centrifuge to separate the solid and liquid, set the centrifuge speed to 8000 r / min, centrifuge for 15 min, and collect the supernatant; (4) spray drying the supernatant obtained in step (3) under the conditions of an inlet temperature of 120° C., an outlet temperature of 56° C. to 60° C., a fan speed of 97%, a peristaltic pump speed of 30%, and a needle-passing interval of 5 s to obtain a chamacha alkaline extract having hypoxia-resistant activity.

2. The method for preparing a chamacha alkali extract having hypoxia-resistant activity according to claim 1, characterized in that The polysaccharide content of the hypoxia-resistant Chamaglossum alkaloid extract is 50.1%-56.34%, the protein content is 0.1%-0.5%, the polyphenol content is 0.32%-0.47%, and the flavonoid content is 0.05%-0.15%.

3. The method for preparing a chamacha alkali extract having hypoxia-resistant activity according to claim 1, characterized in that After feeding chamacarpine extract, the survival time of mice in the medium-dose group in the normal pressure closed hypoxia experiment was extended by up to 13.02% compared with the blank group. In the sodium nitrite poisoning experiment, the survival time of the low-dose group was extended by up to 3.91% compared with the blank group. In the experiment of reducing the malondialdehyde (MDA) content in the heart, lung and liver tissues of the high-dose group, the maximum values ​​were 21.95%, 32.0% and 45.16% respectively, and in the experiment of increasing the superoxide dismutase (SOD) content, the maximum values ​​were 37.47%, 36.52% and 20.56% respectively.

4. A chamacarpa alkaloid extract having hypoxia-resistant activity prepared by the preparation method according to any one of claims 1 to 3.

5. The use of the chamacha alkali extract having hypoxia-resistance activity according to claim 4, characterized in that The chamacarpa alkali extract is used for preparing compressed candies and biscuits as hypoxia-resistant functional foods.

Images