Plant mixed extract capable of efficiently activating anti-hypoxia activity of cardiac cells as well as preparation method and application of plant mixed extract

Abstract

The invention discloses a plant composition capable of efficiently activating anti-hypoxia activity of cardiac cells and a preparation method thereof.The preparation method comprises the steps that cardamine violifolia and rhodiola rosea are smashed and then mixed and pulped, water is added, pectinase, cellulase and hemicellulase are added for ultrasonic enzymolysis after high-pressure homogenization, centrifugal separation is performed, and a mixture is obtained; and after filtering, freezing and drying the supernate to obtain a mixed extract of cardamine violifolia and rhodiola rosea. According to the method, plant cell walls are crushed firstly through ultrahigh pressure extraction, functional components in cells are released, the functional components are dissolved in an extracting solution to the maximum extent under the action of enzymolysis and ultrasound, meanwhile, it can be guaranteed that chemical bonds of the functional components are not damaged, and the structural integrity and biological activity of the functional components are reserved to the maximum extent. The cardamine violifolia and the rhodiola rosea in the mixed extract have a synergistic effect, so that the taste of the product can be improved and the purpose of reducing the cost can be achieved under the condition that the use amount and the use period of the rhodiola rosea can be reduced.

Description

Technical Field

[0001] This invention belongs to the field of extraction and preparation technology of plant functional component compositions, specifically relating to a plant mixed extract that efficiently activates the anti-hypoxia activity of heart cells, its preparation method and application. Background Technology

[0002] Oxygen is a crucial substance for the body to obtain energy. Insufficient oxygen supply to tissues or impaired oxygen utilization leads to hypoxia, causing a series of changes in the body and even various acute and chronic diseases. Hence the saying that hypoxia is the root of all diseases. Hypoxia causes the body to produce a large amount of reactive oxygen species (ROS), resulting in an imbalance between the oxidative and antioxidant systems. A large number of free radicals attack the body's DNA, lipids, and proteins, causing damage to the cardiovascular and nervous systems. Oxidative stress is the main cause of hypoxic damage.

[0003] In high-altitude areas, especially high-altitude regions, prolonged hypoxia can have physiological effects on the respiratory, cardiovascular, and nervous systems. Acute exposure to high-altitude hypoxia can impair the body's oxygen supply capacity and exercise endurance, leading to fatigue. In severe cases, it can even cause loss of work capacity, and the decline in physical performance becomes more significant with increasing altitude. Hypoxia prevents the body from obtaining sufficient oxygen, reducing the metabolic capacity of various tissues and organs, thus causing fatigue. Furthermore, hypoxia can lead to insufficient oxygen supply to the brain, affecting the normal function of the nervous system and further exacerbating fatigue.

[0004] In recent years, more and more people working and traveling have entered high-altitude areas. However, most people who quickly enter the plateau will experience oxidative stress due to their inability to adapt to the hypoxic environment, which can damage the heart, lungs, brain and other tissues and organs. In severe cases, it can lead to a series of diseases such as pulmonary edema and cerebral edema.

[0005] With the increasing number of people traveling to and working in high-altitude areas, improving the physical function and alleviating fatigue in these individuals has become a pressing issue in high-altitude medicine. Acetazolamide, a commonly used carbonic anhydrase inhibitor, enhances the body's adaptability by increasing respiratory rate and is currently a primary drug for preventing altitude sickness (AMS). Under acute hypoxia, acetazolamide can reduce intraerythrocyte CO2 concentration and promote the release of oxygen from oxyhemoglobin. However, its clinical application has been less than satisfactory; acetazolamide can even reduce physical performance and increase leg fatigue under hypoxic conditions. Sildenafil is another drug used to treat cardiovascular diseases. It works by inhibiting phosphodiesterase-5 (PDE5) enzyme, increasing cyclic guanosine monophosphate (cGMP) levels, dilating pulmonary blood vessels, lowering pulmonary artery pressure, and improving pulmonary blood flow and oxygenation. This can reduce hypoxic pulmonary hypertension during rest and exercise, while maintaining gas exchange and systemic blood pressure. Sildenafil was the first drug to increase exercise capacity in areas with severe hypoxia at sea level and high altitudes. However, sildenafil can cause hypotension and adverse cardiac reactions, as well as adverse effects on the digestive, respiratory, and nervous systems. Long-term use can be very harmful to the body. Therefore, developing supplements containing plant-based functional components that enhance the hypoxia resistance of heart cells can not only effectively reduce the incidence of acute altitude sickness but also enhance the body's altitude adaptability and improve work capacity.

[0006] Traditional Chinese medicine has a long history of clinical application in preventing and treating altitude sickness and hypoxia-ischemia. Single herbs such as ginseng and rhodiola rosea have been proven to exert anti-hypoxia effects by reducing oxidative stress, inhibiting the expression of inflammatory factors, and suppressing cell apoptosis. For example, rhodiola rosea is a traditional Chinese medicine that is both food and medicine; studies have shown that its active ingredients can prevent and alleviate hypoxia-related damage. Rhodiola rosea contains active ingredients such as rhodioloside, which can stimulate bone marrow hematopoiesis, promote erythrocyte production, and increase hemoglobin levels in the blood, thereby improving the blood's oxygen-carrying capacity. In low-oxygen environments (such as at high altitudes), the blood can more efficiently transport oxygen to all tissues, alleviating hypoxia symptoms. However, in most cases, rhodiola rosea used alone has a relatively weak effect and requires supplementation well in advance (7-15 days), often requiring larger doses and resulting in higher costs. Therefore, based on the anti-hypoxia capabilities of rhodiola rosea, it is necessary to develop a plant-based mixed extract to enhance the anti-hypoxia capabilities of single herbs. Summary of the Invention

[0007] To address the aforementioned problems, the purpose of this invention is to provide a plant-based mixed extract that effectively activates the anti-hypoxia activity of cardiac cells and its preparation method, thereby solving the problems of poor hypoxia improvement and slow action when using Rhodiola rosea extract alone.

[0008] To address the above problems, this invention first provides a method for preparing a plant-based mixed extract that efficiently activates the anti-hypoxia activity of cardiac cells, comprising the following steps:

[0009] (1) Wash the fresh violet leaves and water chestnuts, wash the rhodiola rosea, chop them, mix them together, make a paste, add water, and then use an ultra-high pressure homogenizer to break the cell wall and extract the extract.

[0010] (2) Adjust the pH of the mixture obtained in step (1) to 7-8, add pectinase, cellulase and hemicellulase, and perform enzymatic extraction under ultrasonic treatment;

[0011] (3) Centrifuge the mixture after enzymatic hydrolysis in step (2), take the supernatant, filter it, and freeze-dry the filtrate to obtain a mixed extract of Viola yedoensis and Rhodiola rosea.

[0012] In one embodiment of the present invention, in step (1), the mixing ratio of the two is 1:1 to 12, preferably 1:2 to 12, and more preferably 1:2 to 10.

[0013] In one embodiment of the present invention, in step (1), the mass ratio of the pulped mixture to water is 1:5 to 20.

[0014] In one embodiment of the present invention, in step (1), the high-pressure homogenization pressure is 150-200 MPa, the temperature is controlled between 20-30°C, and the high-pressure homogenization time is 1-5 min.

[0015] In one embodiment of the present invention, in step (2), the total amount of pectinase, cellulase and hemicellulase added is 0.5-2%, wherein the mass ratio of pectinase, cellulase and hemicellulase is 1:1:1.

[0016] In one embodiment of the present invention, in step (2), the activity of pectinase is 21,000 U / mL, the activity of cellulase is 3,500 U / g, and the activity of hemicellulase is 50,000 U / g.

[0017] In one embodiment of the present invention, in step (2), the temperature during enzymatic extraction is 50-55℃, the ultrasonic frequency is greater than 20-40kHz, the ultrasonication is performed for 10-30 minutes, and enzymatic extraction is continued for 0.5-4 hours after ultrasonication.

[0018] In one embodiment of the present invention, in step (3), the temperature during centrifugation is 0-4℃, the centrifugation speed is 2000-3000r / min, and the centrifugation time is 2-5min.

[0019] In one embodiment of the present invention, in step (3), filtration is performed using an 800nm ​​membrane to remove small-molecule inorganic salts and heavy metal ions (sodium, cadmium, arsenic, etc.).

[0020] In one embodiment of the present invention, in step (3), the freeze-drying process is first carried out at -20°C for 24 hours, and after it is solidified, it is placed in a vacuum freeze dryer for drying (vacuum degree 30Mpa, temperature -40°C, time 24 hours) to obtain a mixed extract of Viola yedoensis and Rhodiola rosea.

[0021] The present invention also provides a mixed extract of Viola yedoensis and Rhodiola rosea prepared according to the above method.

[0022] The present invention also provides the application of the above-mentioned mixed extract of Viola yedoensis and Rhodiola rosea in food, health products and pharmaceuticals.

[0023] Beneficial effects:

[0024] (1) Viola yedoensis is a cruciferous plant rich in protein, polysaccharides, flavonoids and other plant functional components. In 2021, the National Health Commission officially approved it as a leafy vegetable, making it a high-quality food ingredient. This invention involves pulverizing Viola yedoensis and Rhodiola rosea, mixing them, and then extracting the mixed extract under high pressure homogenization and enzymatic hydrolysis. In the mixed extract of this invention, Viola yedoensis and Rhodiola rosea work synergistically to rapidly and efficiently enhance the composition's anti-hypoxia ability. This reduces the amount and duration of Rhodiola rosea use, improves product taste, and lowers costs. It is safer than Western medicine ingredients and greatly reduces toxic side effects.

[0025] (2) This invention employs ultra-high pressure extraction to first break the plant cell wall and release the functional components inside the cell; then, it uses a compound enzyme to enzymatically digest the cellulose, hemicellulose, pectin and other substances that are bound to the functional components to further release the functional components; at the same time, it uses ultrasonic cavitation effect to maximize the dissolution of the functional components in the extract. The above steps are progressive, which can achieve the maximum separation and extraction of functional components while ensuring that the chemical bonds of the functional components are not damaged, thus preserving their structural integrity and biological activity to the greatest extent.

[0026] (3) When the extracts of Viola yedoensis and Rhodiola rosea are used alone, their effect on activating the anti-hypoxia activity of heart cells is not particularly obvious. When the extracts of the two plants are extracted separately and mixed in a certain proportion, the two extracts interact and have a certain effect on improving the anti-hypoxia ability. When Viola yedoensis and Rhodiola rosea are mixed together to extract the plant extract mixture, the anti-hypoxia ability of Rhodiola rosea extract can be significantly enhanced. Under the same effect, the addition of Viola yedoensis can reduce the amount of Rhodiola rosea used and also greatly shorten the use cycle of Rhodiola rosea (7-15 days when Rhodiola rosea is used alone). When Viola yedoensis and Rhodiola rosea are used together, only 5-7 days are needed, thereby reducing the product cost. It is a high-quality health food plant compound raw material. Detailed Implementation

[0027] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several changes and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0028] The pectinase used in the embodiments and comparative examples of this invention is from Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd., with an enzyme activity of 21,000 U / mL; the cellulase is from Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd., with an enzyme activity of 3,500 U / g; and the hemicellulase is from Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd., with an enzyme activity of 50,000 U / g.

[0029] Example 1

[0030] A method for preparing a plant composition that efficiently activates the anti-hypoxia activity of cardiac cells includes the following steps:

[0031] (1) Wash the fresh violet leaves and water chestnuts, wash the rhodiola rosea, chop them and mix them at a mass ratio of 1:10. Pulp the mixture and add purified water at a material-to-liquid ratio of 1:10. Then, use an ultra-high pressure homogenizer to break the cell wall and extract the mixture. The homogenization pressure is 150 MPa, the homogenization time is 3 min, and the temperature is controlled between 20-30℃. After that, adjust the pH value to 7.5 and add pectinase, cellulase and hemicellulase (the amount added is 0.6% of the weight of the fresh vegetables, and the ratio of the three enzymes is 1:1:1) for enzymatic extraction. The extraction temperature is 50℃, the ultrasonic frequency is 25 kHz, the ultrasonic treatment is 10 min, and then the enzymatic treatment is carried out for 1 h.

[0032] (2) After the enzymatic extraction is completed, high-speed low-temperature centrifugation is performed. The centrifuge temperature is 4℃, the centrifugation speed is 3000r / min, and the centrifugation time is 3min. The supernatant is then collected.

[0033] (3) After filtering the supernatant obtained in step (2) with an 800nm ​​membrane to remove small molecule inorganic salts and heavy metal ions (sodium, cadmium, arsenic, etc.), the filtrate is freeze-dried. First, it needs to be frozen at -20℃ for 24 hours, and after it is solidified, it is placed in a vacuum freeze dryer for drying (vacuum degree 30Mpa, temperature -40℃, time 24h) to obtain the mixed extract of Viola yedoensis and Rhodiola rosea.

[0034] Example 2

[0035] The difference between Example 2 and Example 1 is that the mass ratio of Viola yedoensis and Rhodiola rosea in step (1) is 1:2.

[0036] A method for preparing a plant composition that efficiently activates the anti-hypoxia activity of cardiac cells includes the following steps:

[0037] (1) Wash the fresh violet leaves and water chestnuts thoroughly, wash the Rhodiola rosea thoroughly, chop them and mix them at a mass ratio of 1:2. Add purified water at a material-to-liquid ratio of 1:10 and use an ultra-high pressure homogenizer for cell wall breaking extraction. The homogenization pressure is 150 MPa, the homogenization time is 4 min, and the temperature is controlled between 20-30℃. After the ultra-high pressure homogenization is completed, adjust the pH value to 7.5 and add pectinase, cellulase and hemicellulase (the amount added is 0.6% of the weight of the fresh vegetables, and the ratio of the three enzymes is 1:1:1). The extraction temperature is 50℃, the ultrasonic frequency is 25 kHz, and the ultrasonic treatment is 10 min. Then, enzymatic hydrolysis is performed for 1 h.

[0038] (2) After the enzymatic extraction is completed, high-speed low-temperature centrifugation is performed. The centrifuge temperature is 4℃, the centrifugation speed is 3000r / min, and the centrifugation time is 3min. The supernatant is then collected.

[0039] (3) After filtering the supernatant obtained in step (2) with an 800nm ​​membrane to remove small molecule inorganic salts and heavy metal ions (sodium, cadmium, arsenic, etc.), the filtrate is freeze-dried. First, it needs to be frozen at -20℃ for 24 hours, and after it is solidified, it is placed in a vacuum freeze dryer for drying (vacuum degree 30Mpa, temperature -40℃, time 24h) to obtain the mixed extract of Viola yedoensis and Rhodiola rosea.

[0040] Example 3

[0041] The difference between Example 3 and Example 1 is that the mass ratio of Viola yedoensis and Rhodiola rosea in step (1) is 1:15.

[0042] A method for preparing a plant composition that activates the anti-hypoxia activity of heart cells includes the following steps:

[0043] (1) Wash the fresh violet leaves and water chestnuts thoroughly, wash the Rhodiola rosea thoroughly, chop them and mix them at a mass ratio of 1:15. Add purified water at a material-to-liquid ratio of 1:10 and use an ultra-high pressure homogenizer for cell wall breaking extraction. The homogenization pressure is 150 MPa, the homogenization time is 3 min, and the temperature is controlled between 20-30℃. After the ultra-high pressure homogenization is completed, adjust the pH value to 7.5 and add pectinase, cellulase and hemicellulase (the amount added is 0.6% of the weight of the fresh vegetables, and the ratio of the three enzymes is 1:1:1). The extraction temperature is 50℃, the ultrasonic frequency is 25 kHz, and the ultrasonic treatment is 10 min. Then, enzymatic hydrolysis is performed for 1 h.

[0044] (2) After the enzymatic extraction is completed, high-speed low-temperature centrifugation is performed. The centrifuge temperature is 4℃, the centrifugation speed is 3000r / min, and the centrifugation time is 3min. The supernatant is then collected.

[0045] (3) After filtering the supernatant obtained in step (2) with an 800nm ​​membrane to remove small molecule inorganic salts and heavy metal ions (sodium, cadmium, arsenic, etc.), the filtrate is freeze-dried. First, it needs to be frozen at -20℃ for 24 hours, and after it is solidified, it is placed in a vacuum freeze dryer for drying (vacuum degree 30Mpa, temperature -40℃, time 24h) to obtain the mixed extract of Viola yedoensis and Rhodiola rosea.

[0046] Example 4

[0047] The difference between Example 4 and Example 1 is that the mass ratio of Viola yedoensis and Rhodiola rosea in step (1) is 1:5.

[0048] A method for preparing a plant composition that efficiently activates the anti-hypoxia activity of cardiac cells includes the following steps:

[0049] (1) Wash the fresh violet leaves and water chestnuts thoroughly, wash the Rhodiola rosea thoroughly, chop them and mix them at a mass ratio of 1:5. Add purified water at a material-to-liquid ratio of 1:10 and use an ultra-high pressure homogenizer for cell wall breaking extraction. The homogenization pressure is 150 MPa, the homogenization time is 4 min, and the temperature is controlled between 20-30℃. After the ultra-high pressure homogenization is completed, adjust the pH value to 7.5 and add pectinase, cellulase and hemicellulase (the amount added is 0.8% of the weight of the fresh vegetables, and the ratio of the three enzymes is 1:1:1). The extraction temperature is 50℃, the ultrasonic frequency is 25 kHz, and the ultrasonic treatment is 10 min. Then, enzymatic hydrolysis is performed for 1 h.

[0050] (2) After the enzymatic extraction is completed, high-speed low-temperature centrifugation is performed. The centrifuge temperature is 4℃, the centrifugation speed is 3000r / min, and the centrifugation time is 3min. The supernatant is then collected.

[0051] (3) After filtering the supernatant obtained in step (2) with an 800nm ​​membrane to remove small molecule inorganic salts and heavy metal ions (sodium, cadmium, arsenic, etc.), the filtrate is freeze-dried. First, it needs to be frozen at -20℃ for 24 hours, and after it is solidified, it is placed in a vacuum freeze dryer for drying (vacuum degree 30Mpa, temperature -40℃, time 24h) to obtain the mixed extract of Viola yedoensis and Rhodiola rosea.

[0052] Comparative Example 1

[0053] The difference between Comparative Example 1 and Example 1 is that fresh violet leaves and water chestnuts are not added in step (1).

[0054] A method for preparing a plant extract that activates the anti-hypoxia activity of cardiac cells includes the following steps:

[0055] (1) After cleaning Rhodiola rosea, crush it and add purified water (the ratio of material to liquid is 1:10). Use an ultra-high pressure homogenizer to break the cell wall and extract it. The homogenization pressure is 150 MPa, the homogenization time is 3 min, and the temperature is controlled between 20-30℃.

[0056] (2) After the ultra-high pressure homogenization is completed, the pH value is adjusted to 7.5, and pectinase, cellulase and hemicellulase are added (the amount added is 0.6% of the weight of fresh vegetables, and the ratio of the three enzymes is 1:1:1). The extraction temperature is 50℃, the ultrasonic frequency is 25KHz, the ultrasonic treatment is 10 minutes, and then the enzymatic hydrolysis is performed for 1 hour.

[0057] (3) After the enzymatic extraction is completed, high-speed low-temperature centrifugation is performed. The centrifuge temperature is 4℃, the centrifugation speed is 3000r / min, and the centrifugation time is 3min. The supernatant is collected. After filtering with an 800nm ​​membrane to remove small molecule inorganic salts and heavy metal ions (sodium, cadmium, arsenic, etc.), the filtrate is freeze-dried. First, it needs to be frozen at -20℃ for 24h. After it is frozen solid, it is placed in a vacuum freeze dryer for drying (vacuum degree 30Mpa, temperature -40℃, time 24h) to obtain Rhodiola rosea extract.

[0058] Comparative Example 2

[0059] The difference between Comparative Example 2 and Example 1 is that Rhodiola rosea is not added in step (1).

[0060] A method for preparing a plant extract that activates the anti-hypoxia activity of cardiac cells includes the following steps:

[0061] (1) Clean the fresh Viola yedoensis and water chestnut, chop them and pulp them. Add purified water at a ratio of 1:10 and use an ultra-high pressure homogenizer to break the cell wall for extraction. The homogenization pressure is 160 MPa and the temperature is controlled at 20℃. After completion, adjust the pH value to 7.5 and add pectinase, cellulase and hemicellulase (the amount added is 0.6% of the weight of the fresh vegetables, and the ratio of the three enzymes is 1:1:1). The extraction temperature is 50℃, the ultrasonic frequency is 25 kHz, and the ultrasonic treatment is 10 min. Then, enzymatic hydrolysis is performed for 1 h.

[0062] (2) After the enzymatic extraction is completed, high-speed low-temperature centrifugation is performed. The centrifuge temperature is 4℃, the centrifugation speed is 3000r / min, and the centrifugation time is 3min. The supernatant is then collected.

[0063] (3) Use an 800nm ​​membrane to filter out small molecules of inorganic salts and heavy metal ions (sodium, cadmium, arsenic, etc.). Freeze-dry the filtrate. First, freeze at -20℃ for 24 hours. After it is frozen solid, place it in a vacuum freeze dryer for drying (vacuum degree 30Mpa, temperature -40℃, time 24h) to obtain Viola yedoensis extract.

[0064] Comparative Example 3

[0065] A hypoxia-resistant plant extract composition comprising 1 part of Viola yedoensis extract and 1 part of Rhodiola rosea extract, which are mixed evenly.

[0066] The preparation method of Viola yedoensis extract was the same as that of Comparative Example 2, and the preparation method of Rhodiola rosea extract was the same as that of Comparative Example 1.

[0067] Comparative Example 4

[0068] The difference between Comparative Example 4 and Comparative Example 3 is that the composition includes 1 part of Viola yedoensis extract and 5 parts of Rhodiola rosea extract, which are mixed evenly.

[0069] Comparative Example 5

[0070] The difference between Comparative Example 5 and Comparative Example 3 is that the composition contains 1 part of Viola yedoensis extract and 10 parts of Rhodiola rosea extract, which are mixed evenly.

[0071] Zebrafish experimental method:

[0072] Healthy 4dpf wild-type zebrafish were randomly selected and placed in 24-well plates, with 10 fish per well. The Rhodiola rosea extract, Viola yedoensis extract, and Rhodiola rosea mixed extract were diluted to concentrations of 10, 25, 50, and 125 μg / mL and added to the prepared 24-well plates, respectively. The normal control group and the hypoxia model group were treated with fish tank water for 48 hours, and the treatment solution was changed daily during the treatment period.

[0073] Zebrafish hypoxia modeling and movement behavior tracking:

[0074] After the zebrafish pre-protection treatment (drug administration) was completed, each group of juvenile fish was transferred to a custom 24-well plate, one fish per well. The original culture medium was removed, and a fresh 8 mg / mL sodium sulfite solution was prepared. The normal control group was treated with 1 mL of normal fish-raising water, while the hypoxia model group and each sample treatment group were treated with 1 mL of sodium sulfite solution for 20 min to induce hypoxia modeling. Then, the movement images of the zebrafish juveniles within 10 min after hypoxia were captured using a zebrafish two-dimensional tracking system. The movement trajectory of each group of zebrafish was recorded, and the movement trajectory, time and other indicators of each group of zebrafish were quantified using the accompanying advanced video image analysis and processing program. The movement distance and movement-resting time of each group of juvenile fish were calculated and statistically analyzed. Resting time refers to the time spent at rest excluding movement.

[0075] Statistics on the number of zebrafish that survived oxygen deprivation:

[0076] When zebrafish are deprived of oxygen, they will exhibit symptoms such as coma, floating to the surface, rolling to their side, and bending of the trunk. At this time, each group of zebrafish hypoxia models is removed from the sodium sulfite solution and placed in normal fish farming water for recovery treatment. After a period of time, the number of surviving zebrafish in each group is calculated to evaluate the anti-hypoxia effect of the samples.

[0077] Zebrafish experiments were conducted using extracts or compositions prepared according to the examples and comparative examples, and the results are shown in Table 1. The normal group consisted of zebrafish treated with normal fish-raising water, the hypoxic group consisted of zebrafish treated with fish-raising water after hypoxia modeling, and the rest were experimental groups. The experimental groups used extract compositions or extracts prepared according to the examples or comparative examples to treat the hypoxic zebrafish. Experimental groups 1-4 corresponded to Examples 1-4, respectively, using 50 μg / mL of the extracts from the examples to treat hypoxic zebrafish. Experimental groups 5-6 corresponded to Comparative Examples 1-2, respectively, using 50 μg / mL of the extracts from the comparative examples to treat hypoxic zebrafish. Experimental group 7 used a mixture of 50 μg / mL sodium selenite and 50 μg / mL Rhodiola rosea extract to treat hypoxic zebrafish. Experimental groups 8-10 corresponded to Comparative Examples 3-5, using mixtures of 50 μg / mL Viola yedoensis and Rhodiola rosea extracted separately and then mixed in different proportions to treat hypoxic zebrafish. After treatment, the zebrafish's movement time, resting time, and movement distance were observed, and the contents of LDH lactate dehydrogenase, citrate synthase (CS), and hypoxia-inducible factor-1α (HIF-1α) were detected.

[0078] Lactate dehydrogenase (LDH) detection method: After the experiment, the experimental fish used to test for hypoxia and asphyxiation were prepared into a physiological saline homogenate (diluted 10 times) in a low-temperature environment. The supernatant was collected by centrifugation, diluted 20 times, and the LDH assay kit was added according to the steps. The reaction was allowed to proceed for 15 minutes, and the absorbance was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The activity of LDH was calculated using a standard curve.

[0079] Citrate synthase (CS) assay method: After the experiment, the experimental fish used to determine hypoxia and asphyxiation were prepared into a physiological saline homogenate (diluted 10 times) in a low-temperature environment. The supernatant was collected by centrifugation and diluted 20 times. The citrate synthase (CS) assay kit was added according to the steps. The reaction was allowed to proceed for 15 minutes. The absorbance was measured at a wavelength of 412 nm using an ELISA reader. The activity of lactate dehydrogenase was calculated using a standard curve.

[0080] Hypoxia-inducible factor-1α (HIF-1α) content: determined by ELISA. (Reference: Ma Zhihui. Study on the anti-hypoxia effects of American ginseng and its main active ingredients based on zebrafish hypoxia model [D]. Shandong Medical University, 2023)

[0081] Table 1. Experimental results of zebrafish in the normal group, hypoxia group, and experimental group.

[0082]

[0083] Table 1 shows the movement time and distance, resting time, and levels of LDH enzyme, CS enzyme, and hypoxia-inducible factor-1α in zebrafish from the normal group, hypoxia group, and experimental group. As can be seen from Table 1, when hypoxic zebrafish were treated with 50 μg / mL Rhodiola rosea extract alone, their movement distance was 45.12 cm; when treated with 50 μg / mL Viola yedoensis extract alone, their movement distance was 44.38 cm. Compared to Rhodiola rosea extract alone, the movement time, resting time, and movement distance were similar. Furthermore, the levels of lactate dehydrogenase, citrate synthase, and hypoxia-inducible factor-1α were very similar, indicating that both methods, when used alone, had only moderate effectiveness against hypoxia. Compared with the hypoxic group, the use of 50 μg / mL Rhodiola rosea extract or Viola yedoensis extract can effectively increase the movement time and distance of zebrafish, and the levels of LDH enzyme, CS enzyme and hypoxia-inducible factor in their bodies are significantly reduced, indicating that both Rhodiola rosea extract and Viola yedoensis extract have good anti-hypoxia ability.

[0084] Experiments 1, 2, and 4 all used a mixed extract of Rhodiola rosea and Viola yedoensis to treat the hypoxic group. The results showed that adding Viola yedoensis to the extract in groups 1, 2, and 4 significantly increased the movement time and distance of zebrafish, while reducing the corresponding enzyme content. Compared to experimental groups 5-6, which were treated with Rhodiola rosea and Viola yedoensis extracts alone, the concentrations of the extracts used in experiments 1, 2, and 4 were the same. At the same concentration, the movement time of zebrafish in groups 1, 2, and 4 was significantly longer than that in groups 5-6, indicating a synergistic effect between the two extracts in the mixed extract.

[0085] With the increase of the amount of violet leaf fragments and watercress added, the movement time and distance of zebrafish increased significantly. When the mass ratio of violet leaf fragments and watercress to Rhodiola rosea was 1:10, the movement time of zebrafish was already close to that of the normal group. With the continued increase of the amount of violet leaf fragments and watercress added, the movement time, movement distance, and enzyme content of zebrafish became closer and closer to the normal group. However, the increase in the movement time and distance of zebrafish slowed down significantly, indicating that when the mass ratio of violet leaf fragments and watercress to Rhodiola rosea was 1:2 to 10, the two had played an optimal synergistic role.

[0086] Furthermore, after using a mixed extract of Viola yedoensis and Rhodiola rosea, regardless of the mass ratio of 1:10, 1:5, or 1:2, the content of lactate dehydrogenase in zebrafish was significantly reduced, while the content of citrate synthase was significantly increased, and the content of hypoxia-inducible factor-1α was significantly reduced. This demonstrates that the mixed extract of Viola yedoensis and Rhodiola rosea has a stronger antioxidant capacity than Rhodiola rosea extract alone, indicating a significant synergistic effect between the two. The use of Viola yedoensis significantly enhances the hypoxia resistance of Rhodiola rosea. When using a 1:15 ratio of Viola yedoensis and Rhodiola rosea, the changes in the above indicators were not particularly significant, therefore the synergistic effect of a 1:15 ratio of Viola yedoensis and Rhodiola rosea is limited.

[0087] The above comparative experiments all show that when the ratio of Viola yedoensis to Rhodiola rosea is 1:2, 1:5, and 1:10, increasing the mixed extract of Viola yedoensis can significantly increase the movement distance and movement time of zebrafish. It can also enhance the anti-hypoxia ability of cells and organisms by regulating the content of lactate dehydrogenase, citrate synthase and hypoxia-inducible factor. This indicates that the mixture of Viola yedoensis and Rhodiola rosea can enhance the movement activity of zebrafish under hypoxic conditions and enhance their anti-hypoxia ability.

[0088] For experimental group 7, the increase of sodium selenite had virtually no positive effect on the hypoxia resistance of Rhodiola rosea extract itself. In fact, the hypoxia resistance of Rhodiola rosea extract itself decreased to some extent with the increase of sodium selenite concentration. The possible reason is that sodium selenite does not produce a synergistic effect on Rhodiola rosea extract. As the concentration of sodium selenite increases, its toxicity to zebrafish also increases to some extent, resulting in a decrease in the zebrafish's resistance to hypoxia.

[0089] Experimental groups 8-10 used extracts of Viola yedoensis and Rhodiola rosea extracted separately, mixed in ratios of 1:2, 1:5, and 1:10 to treat zebrafish in the hypoxia group. The results showed that the effect was better than the treatment groups treated with Viola yedoensis and Rhodiola rosea alone, but not as good as the effect of the mixed extract of Viola yedoensis and Rhodiola rosea. Therefore, it may prove that Viola yedoensis and Rhodiola rosea produced a synergistic effect when mixed and extracted in this invention, thereby enhancing the anti-hypoxia ability of the combination of the two.

[0090] H9C2 cardiomyocyte hypoxia test:

[0091] H9C2 cardiomyocytes were cultured at 37°C with 5% CO2 in DMEM medium containing 10% fetal bovine serum. When the cells reached 70-80% of the culture dish area, they were passaged. The culture medium was changed every 3 days. Cells in the logarithmic growth phase were used for subsequent experiments.

[0092] Cell count adjusted to cell concentration of 1×10⁻⁶ 4 H9C2 cells were seeded at a density of 1 mL / well in 24-well plates. When the confluence of H9C2 cells reached 70-80% after culture at 37℃ and 5% CO2, the culture conditions were changed to 37℃, 95% N2 + 5% CO2. Hypoxic culture times were sequentially 0 (Control), 6, 12, 24, and 48 h. LDH and SOD levels were measured to determine the optimal hypoxic culture time of 24-48 h. Cell counts were performed, and the cell density was adjusted to 1 × 10⁻⁶ cells / well. 4Cells were seeded at a concentration of 100 μg / mL in 96-well plates, 200 μL per well. After 24 h of seeding, when cell adhesion reached over 80% under an inverted phase-contrast microscope, drug intervention was initiated. Experimental groups 1-4 were treated with a 50 μg / mL mixture of extracts from Examples 1-4 at 0.2 mL, at 37°C, 95% N2 + 5% CO2 for 24 h. Experimental groups 5-6 were treated with a 50 μg / mL extract from Comparative Examples 1-2 at 0.2 mL, at 37°C, 95% N2 + 5% CO2 for 24 h. The subjects were treated with 5% CO2 for 48 hours. Experimental group 7 used a mixture of 50 μg / mL sodium selenite and 50 μg / mL Rhodiola rosea extract, added at 0.2 mL, and treated at 37℃ with 95% N2 + 5% CO2 for 48 hours. Experimental groups 8-10 corresponded to groups 3-5 in proportion, using a mixture of 50 μg / mL Viola yedoensis extract and Rhodiola rosea extract, added at 0.2 mL, and treated at 37℃ with 95% N2 + 5% CO2 for 48 hours. The normal control group received no treatment, and the hypoxic groups received no drug intervention.

[0093] After 24 hours of drug intervention, the culture medium was discarded, and the cells were washed three times with phosphate-balanced saline (PBS) at 37°C. Then, 10 μL of thiazolyl blue (MTT) and 5 mg / mL of thiazolyl blue (MTT) were added sequentially, and the cells were cultured for another 4 hours. The supernatant was discarded, and 100 μL of dimethyl fluorescein (DMSO) was added. The cells were then shaken on a shaker for 10 minutes to fully dissolve the crystals. The optical density was measured at 490 nm using a microplate reader, and the corresponding levels of LDH (lactic acid dehydrogenase), SOD (superoxide dismutase), and malondialdehyde (MDA) were calculated.

[0094] Table 2. Content of corresponding substances in cells of normal group, hypoxia group and experimental group

[0095]

[0096]

[0097] As shown in Table 2, under hypoxic conditions, LDH and MDA levels in H9C2 cardiomyocytes significantly increased. Adding Rhodiola rosea extract to hypoxic cells reduced LDH and MDA levels to some extent, but the effect was not very significant. Adding Viola yedoensis extract alone reduced LDH and MDA levels to some extent, but the reduction was not significant. Adding a mixed extract of Viola yedoensis and Rhodiola rosea (experimental group 3, ratio 1:15) showed a significant improvement compared to using Rhodiola rosea extract alone. The initial sample showed a slight decrease in LDH and MDA, but the effect was not very significant. However, after adding a mixed extract of Viola yedoensis and Rhodiola rosea (in ratios of 1:10, 1:5, and 1:2), even with only 24 hours of drug intervention, the levels of LDH and MDA decreased significantly. The decrease in LDH reached 53.1%, and the decrease in MDA was as high as 36%. This indicates that the mixed extract of Viola yedoensis and Rhodiola rosea significantly and effectively activated the cells' anti-hypoxia ability, restoring the cells to a normal state under hypoxia.

[0098] Under hypoxic conditions, superoxide dismutase (SOD) levels in H9C2 cardiomyocytes significantly decreased. The addition of Rhodiola rosea extract to hypoxic cells resulted in a certain increase in SOD levels, indicating that Rhodiola rosea extract has a certain anti-hypoxic effect. The addition of Viola yedoensis extract alone also resulted in a certain increase in SOD levels, demonstrating that Viola yedoensis extract alone also has some effect. The addition of a mixed extract of Viola yedoensis extract and Rhodiola rosea (at a ratio of 1:15) resulted in a slight increase in SOD levels compared to Rhodiola rosea extract alone, but this increase was not significant. However, the addition of a mixed extract of Viola yedoensis extract and Rhodiola rosea (at ratios of 1:10, 1:5, and 1:2) resulted in a significant increase in SOD levels, even with a drug intervention time of only 24 hours, with the highest increase reaching 109.6%. This indicates that the mixed extract of Viola yedoensis extract and Rhodiola rosea significantly activated the SOD activity of cardiomyocytes, enhanced their anti-hypoxic ability, and restored the cells to a normal state.

[0099] The combined extracts of Viola yedoensis and Rhodiola rosea (in ratios of 1:10, 1:5, and 1:2) achieved effects within 24 hours that surpassed the effects of Rhodiola rosea extract alone after 48 hours. This significantly reduces the onset time required for Rhodiola rosea alone, indicating that Viola yedoensis can synergistically enhance the anti-hypoxia ability of Rhodiola rosea extract. Under the same therapeutic effect, it can reduce the dosage of Rhodiola rosea and significantly shorten its onset time, thus substantially reducing the cost of Rhodiola rosea. Furthermore, the combined extracts of Viola yedoensis and Rhodiola rosea (in ratios of 1:10, 1:5, and 1:2) were more effective than using either extract alone in different ratios (1:10, 1:5, and 1:2). This demonstrates a synergistic effect between the two raw materials during the combined extraction, thereby enhancing the anti-hypoxia activity of the combined extract against cardiomyocytes.

[0100] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A method for preparing a plant-based mixed extract that efficiently activates the anti-hypoxia activity of cardiac cells, characterized in that, Includes the following steps: (1) Wash the fresh Viola yezoensis and Rhodiola rosea, chop them, mix them, grind them into a paste, add water, and then use an ultra-high pressure homogenizer to break the cell wall and extract the paste. The mass ratio of Viola yezoensis and Rhodiola rosea is 1:1 to 12. (2) Adjust the pH of the mixture obtained in step (1) to 7-8, add pectinase, cellulase and hemicellulase, and perform enzymatic extraction under ultrasonic treatment; (3) Centrifuge the mixture after enzymatic hydrolysis in step (2), take the supernatant, filter it, and freeze-dry the filtrate to obtain a mixed extract of Viola yedoensis and Rhodiola rosea.

2. The preparation method according to claim 1, characterized in that, In step (1), the mixing ratio of the two is 1:2 to 10, and the mass ratio of the mixture after pulping to water is 1:5 to 20.

3. The preparation method according to claim 1, characterized in that, In step (1), the high-pressure homogenization pressure is 150-200 MPa, the temperature is controlled between 20-30℃, and the high-pressure homogenization time is 1-5 min.

4. The preparation method according to claim 1, characterized in that, In step (2), the total amount of pectinase, cellulase and hemicellulase added is 0.5-2%, and the mass ratio of pectinase, cellulase and hemicellulase is 1:1:

1.

5. The preparation method according to claim 1, characterized in that, In step (2), the temperature for enzymatic extraction is 50-55℃, the ultrasonic frequency is greater than 20-40kHz, the ultrasonic treatment lasts for 10-30 minutes, and enzymatic extraction continues for 0.5-4 hours after ultrasonic treatment.

6. The preparation method according to claim 1, characterized in that, In step (3), the temperature during centrifugation is 0-4℃, the centrifugation speed is 2000-3000r / min, and the centrifugation time is 2-5min.

7. The preparation method according to claim 1, characterized in that, In step (3), filtration is performed using an 800nm ​​membrane.

8. The preparation method according to claim 1, characterized in that, In step (3), the freeze dryer is first frozen at -20°C for 24 hours, and then placed in a vacuum freeze dryer for 24 hours of drying.

9. A mixed extract of Viola yedoensis and Rhodiola rosea prepared by the preparation method according to any one of claims 1 to 8.

10. The use of the mixed extract of Viola yedoensis and Rhodiola rosea as described in claim 9 in food, health products, and pharmaceuticals.

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