American ginseng fermentation liquor with myocardial ischemia resisting or blood vessel dilating effect as well as preparation method and application of American ginseng fermentation liquor
By fermenting American ginseng, a fermented American ginseng liquid with anti-myocardial ischemia and vasodilatory effects was prepared, which solved the problem of insufficient efficacy of American ginseng and significantly improved its effect in improving myocardial blood supply and lowering blood pressure. Its safety and efficacy were verified through animal experiments.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- TIANJIN UNIV
- Filing Date
- 2025-03-11
- Publication Date
- 2026-05-05
AI Technical Summary
The existing efficacy of American ginseng in dilating blood vessels and resisting myocardial ischemia is relatively weak, and there are few related studies and patents. It is necessary to improve its pharmacodynamic material basis.
Small molecule compounds of American ginseng were modified by fermentation. American ginseng was mixed with MRS medium containing Lactobacillus plantarum ATCC8014 with an OD600 value in the range of 0.5-0.9 and fermented for 24-48 hours to obtain American ginseng fermentation broth. The broth was then inactivated and centrifuged, and finally concentrated under reduced pressure to a specific volume to prepare American ginseng fermentation broth with anti-myocardial ischemia or vasodilatory effects.
American ginseng fermentation liquid significantly enhances the types and efficacy of PPD and PPT type ginsenosides, can improve abnormal ejection fraction caused by myocardial ischemia, lower blood pressure, and has a stronger vasodilatory function. Its safety and efficacy have been verified through animal experiments.
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Figure CN121971501A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of American ginseng extraction and application, specifically relating to an American ginseng fermentation liquid with anti-myocardial ischemia or vasodilatory effects, its preparation method and application. Background Technology
[0003] American ginseng is mainly used in the following areas: (1) Chronic disease management: used to improve fatigue syndrome, enhance immunity, and regulate blood pressure and blood sugar. (2) Adjuvant therapy for tumors: used as an adjuvant drug to reduce the side effects of radiotherapy and chemotherapy and improve the quality of life of patients. (3) Neuropsychiatric diseases: used to relieve anxiety, depression and improve cognitive function. (4) Cardiovascular diseases: used as an adjuvant therapy for cardiovascular diseases. Among these, there are relatively few studies on the prevention and treatment of myocardial ischemia and vasodilators based on blood pressure regulation using American ginseng.
[0004] Cardiovascular disease, as a pathophysiological state, is caused by insufficient blood and oxygen supply to the heart and abnormal blood pressure due to conditions such as coronary artery stenosis, spasm, or embolism. The incidence of cardiovascular disease is increasing year by year, showing a trend towards younger onset and earlier onset. The main goals of treatment for this disease are to improve myocardial blood supply and reduce myocardial oxygen consumption, and to dilate coronary arteries to enhance blood supply. Therefore, vasodilation and prevention of myocardial ischemia are key treatment methods for cardiovascular disease.
[0005] Studies have shown that American ginseng can significantly reduce the activities of myocardial creatine phosphokinase, lactate dehydrogenase, and aspartate aminotransferase in dogs with experimental myocardial infarction. It can also significantly reduce the content of myocardial lipid peroxides and free fatty acids, while increasing the activities of superoxide dismutase and glutathione peroxidase. However, due to the relatively weak pharmacological efficacy of small molecule compounds such as ginsenosides, terpenes, and hydrocarbons in American ginseng, there are few studies and patents related to its vasodilatory and anti-myocardial ischemia effects. Therefore, modifying the small molecule compounds of American ginseng using fermentation methods to alter its pharmacological material basis and enhance its anti-myocardial ischemia and vasodilatory abilities is an urgent problem to be solved. Summary of the Invention
[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a fermented American ginseng liquid with anti-myocardial ischemia or vasodilatory effects.
[0007] The second objective of this invention is to provide a method for preparing American ginseng fermentation liquid with anti-myocardial ischemia or vasodilatory effects.
[0008] A third objective of this invention is to provide the application of the above-mentioned American ginseng fermentation liquid in the preparation of anti-myocardial ischemia products.
[0009] The fourth objective of this invention is to provide the application of the above-mentioned American ginseng fermentation liquid in the preparation of vasodilator products.
[0010] The technical solution of this invention is summarized as follows:
[0011] A method for preparing American ginseng fermentation liquid with anti-myocardial ischemia or vasodilatory effects includes the following steps:
[0012] 1) Add purified water to American ginseng, heat and reflux to extract, filter, combine the filtrates, concentrate the filtrate to obtain concentrated solution;
[0013] 2) Mix the concentrated solution with the OD solution at a volume ratio of 95-99:5-1. 600 Mix with MRS medium containing Lactobacillus plantarum with a pH value in the range of 0.5-0.9, and ferment in a sealed container at 35-39℃ and 10-60 rpm for 24-48 hours to obtain crude fermentation broth; inactivate, centrifuge, and obtain supernatant;
[0014] The Lactobacillus plantarum is Lactobacillus plantarum ATCC8014;
[0015] 3) The supernatant is concentrated under reduced pressure at 50-60℃ to 1 / 3 to 2 / 3 of the supernatant volume to obtain American ginseng fermentation liquid.
[0016] Step 1) is as follows: Add purified water to American ginseng, heat and reflux to extract twice, and the mass of purified water added each time is 8-12 times and 7-9 times the mass of American ginseng, respectively; each extraction lasts 1.5-3 hours, filter, combine the filtrates, and concentrate under reduced pressure at 50-60℃ to a mass equivalent to 4-6 times the mass of American ginseng to obtain a concentrated solution.
[0017] The inactivation steps are as follows: raise the temperature to 55℃-65℃, maintain for 25-35 minutes, then raise the temperature to 70℃-80℃ and maintain for 3-10 minutes.
[0018] The centrifugation speed is 2000-4000 rpm, and the centrifugation time is 10-20 min.
[0019] The above preparation method produces a fermented American ginseng liquid with anti-myocardial ischemia or vasodilatory effects.
[0020] The above-mentioned American ginseng fermentation liquid is used in the preparation of anti-myocardial ischemia products.
[0021] The above-mentioned American ginseng fermentation liquid is used in the preparation of vasodilator products. These products include pharmaceuticals and food products.
[0022] The beneficial effects of this invention are:
[0023] The American ginseng fermentation liquid of this invention contains a richer variety of PPD and PPT type ginsenosides, providing a stronger basis for their effects.
[0024] Experiments have shown that the American ginseng fermentation liquid of this invention can improve ejection fraction and calcium levels caused by myocardial ischemia. 2+ -ATPase and Na + - Abnormalities in ATPase have an anti-myocardial ischemia effect.
[0025] Experiments have shown that the American ginseng fermentation liquid of this invention can lower blood pressure and has the function of dilating blood vessels.
[0026] This invention uses biosafe Lactobacillus plantarum ATCC8014, an edible fungus that will not impose an additional burden on the human body or cause other health problems. Attached Figure Description
[0027] Figure 1 A comparison of the material composition of fermented American ginseng broth and unfermented American ginseng extract.
[0028] Figure 2 A comparison of the types of compounds in fermented and unfermented American ginseng extracts.
[0029] Figure 3 Comparison of ejection fraction in rats with myocardial ischemia treated with fermented American ginseng broth and unfermented American ginseng extract.
[0030] Figure 4 The effects of fermented and unfermented American ginseng extracts on calcium levels in rats with myocardial ischemia. 2+ -ATPase and Na + - Comparison of ATPase regulation.
[0031] Figure 5 This study compares the vasodilatory effects of fermented and unfermented American ginseng extract on rats. Detailed Implementation
[0032] Commercially available MRS culture medium.
[0033] American ginseng is produced in Shandong Province, Liaoning Province, and Jilin Province of China. The embodiments of this invention are based on ginseng produced in Jilin Province, China.
[0034] The Lactobacillus plantarum strain ATCC8014 was purchased from the Guangdong Provincial Microbial Culture Collection Center on February 3, 2023. The strain standard number is GDMCC1.140 and the batch number is 20220927.
[0035] The present invention will be further described below through specific embodiments.
[0036] Example 1
[0037] A method for preparing American ginseng fermentation liquid with anti-myocardial ischemia or vasodilatory effects includes the following steps:
[0038] 1) Add purified water to American ginseng and heat under reflux to extract twice. The mass of purified water added each time is 10 times and 8 times the mass of American ginseng, respectively. Each extraction lasts for 2 hours. Filter the solution, combine the filtrates, and concentrate them under reduced pressure at 55°C to the equivalent of 5 times the mass of American ginseng to obtain a concentrated solution.
[0039] 2) Mix the concentrated solution with OD at a volume ratio of 97:3. 600 Mix with MRS medium containing Lactobacillus plantarum with a pH value in the range of 0.5-0.9, and ferment at 37°C and 30 rpm for 36 hours in a sealed container to obtain crude fermentation broth; inactivate, centrifuge, and obtain supernatant;
[0040] The Lactobacillus plantarum is Lactobacillus plantarum ATCC8014;
[0041] The inactivation steps are as follows: raise the temperature to 60℃, maintain for 30 minutes, then raise the temperature to 75℃ and maintain for 5 minutes.
[0042] The centrifugation speed was 3000 rpm, and the centrifugation time was 15 min;
[0043] 3) The supernatant was concentrated under reduced pressure at 55°C to 1 / 3 of the supernatant volume to obtain American ginseng fermentation liquid 1.
[0044] Example 2
[0045] A method for preparing American ginseng fermentation liquid with anti-myocardial ischemia or vasodilatory effects includes the following steps:
[0046] 1) Add purified water to American ginseng and heat under reflux to extract twice. The mass of purified water added each time is 8 and 7 times the mass of American ginseng, respectively. Each extraction lasts for 3 hours. Filter the solution, combine the filtrates, and concentrate them under reduced pressure at 50°C to a volume equivalent to 4 times the mass of American ginseng to obtain a concentrated solution.
[0047] 2) Mix the concentrated solution with the OD solution at a volume ratio of 95:5. 600 Mix with MRS medium containing Lactobacillus plantarum with a pH value in the range of 0.5-0.9, and ferment at 35℃ and 60 rpm in a sealed container for 48 hours to obtain crude fermentation broth; inactivate, centrifuge, and obtain supernatant;
[0048] The Lactobacillus plantarum is Lactobacillus plantarum ATCC8014;
[0049] The inactivation steps are as follows: raise the temperature to 55℃, maintain for 35 minutes, then raise the temperature to 70℃ and maintain for 10 minutes.
[0050] The centrifugation speed was 2000 rpm, and the centrifugation time was 20 min;
[0051] 3) The supernatant was concentrated under reduced pressure at 50°C to 2 / 3 of the supernatant volume to obtain American ginseng fermentation liquid 2.
[0052] Example 3
[0053] A method for preparing American ginseng fermentation liquid with anti-myocardial ischemia or vasodilatory effects includes the following steps:
[0054] 1) Add purified water to American ginseng and heat under reflux to extract twice. The mass of purified water added each time is 12 and 9 times the mass of American ginseng, respectively. Each extraction lasts 1.5 hours. Filter the solution, combine the filtrates, and concentrate them under reduced pressure at 60°C to a volume equivalent to 6 times the mass of American ginseng to obtain a concentrated solution.
[0055] 2) Mix the concentrated solution with OD at a volume ratio of 99:1. 600 Mix with MRS medium containing Lactobacillus plantarum with a pH value in the range of 0.5-0.9, and ferment at 39°C and 10 rpm for 24 hours in a sealed container to obtain crude fermentation broth; inactivate, centrifuge, and obtain supernatant;
[0056] The Lactobacillus plantarum is Lactobacillus plantarum ATCC8014;
[0057] The inactivation steps are as follows: raise the temperature to 65℃, hold for 25 minutes, then raise the temperature to 80℃ and hold for 3 minutes.
[0058] The centrifugation speed was 4000 rpm and the centrifugation time was 10 min.
[0059] 3) The supernatant was concentrated under reduced pressure at 60°C to 1 / 3 of the supernatant volume to obtain American ginseng fermentation liquid 3.
[0060] Example 4
[0061] The fermented ginseng broth obtained in Example 1 was used as the test sample to study the difference in the material composition of American ginseng due to fermentation.
[0062] Accurately measure 1 mL each of fermented American ginseng broth and unfermented American ginseng extract, evaporate to dryness in a 100℃ water bath, and dissolve in 1 mL of 80% (v / v) methanol aqueous solution. Centrifuge at 13000 rpm for 10 minutes, and analyze using liquid chromatography-mass spectrometry (LC-MS). LC-MS detection conditions: positive and negative ion voltages of 5500 V and 4500 V respectively, detection range 50-1200 Da, detection time 25 minutes, and mobile phase gradient shown in Table 1. The composition of the sample was qualitatively identified by inferring the fragment ion fragmentation pattern.
[0063] Table 1, Mobile Phase Gradient
[0064]
[0065] The test results are as follows:
[0066] After the experiment, the results were analyzed. The types of compounds in the fermented and unfermented American ginseng extracts differed significantly. The unfermented extract contained 132 compounds, while the fermented extract contained 225 compounds (see...). Figure 1 Among these, PPD-type ginsenosides and PPT-type ginsenosides showed the most significant changes; in addition, the number of almost all compounds, including hydrocarbons and triterpenoids, increased (see...). Figure 2 This reflects that the material basis of fermented American ginseng liquid has changed compared to unfermented American ginseng extract, and its medicinal efficacy has been altered.
[0067] The unfermented American ginseng extract is the concentrate obtained in step 1) of Example 1, denoted as concentrate 1. It is further concentrated to 1 / 3 of the volume of concentrate 1 to obtain the unfermented American ginseng extract.
[0068] Example 5
[0069] The American ginseng fermentation broth obtained in Example 1 was used as the test sample to conduct an oral toxicity tolerance test in mice.
[0070] Acute oral toxicity tests in mice were conducted using the maximum tolerated dose method. Twenty-four healthy 8-week-old SPF-grade ICR mice (12 males and 12 females, weighing 18±2g) were selected. The tests were conducted after 7 days of acclimatization.
[0071] Male and female mice were randomly divided into two groups, with 6 males and 6 females in each group, for a total of 12 mice. Each mouse in the American ginseng fermentation liquid group was administered 0.2 mL of American ginseng fermentation liquid by gavage daily, while each mouse in the control group was administered 0.2 mL of distilled water by gavage daily for 14 consecutive days. During the gavage period, mice were fed distilled water and regular rodent food with free access to food. The mice's condition (coat color, diet, mental state, behavior, etc.) and the number of dead mice were recorded during the observation period. Dead mice were promptly dissected, and changes in internal organs were observed, weighed, and recorded. After the experiment, the mice were euthanized by cervical dislocation, and the organs were dissected to observe their color, size, and any visible lesions.
[0072] The test results are as follows:
[0073] The results are shown in Table 1. During the experimental observation period, there was no significant difference in body weight change between the American ginseng fermentation liquid group and the control group. After euthanasia by cervical dislocation, autopsy revealed that the organs were normal in color and size, with no obvious lesions visible to the naked eye. This indicates that the test sample had no effect on mouse growth. The animals maintained normal diet and activity, grew well, showed no signs of poisoning, and there were no deaths. Based on the results of the acute oral toxicity test of the National Food Safety Standard (GB15913.3-2014), the sample was determined to be non-toxic.
[0074] Table 2 Acute oral toxicity in mice (x±SD)
[0075]
[0076] Example 6
[0077] The American ginseng fermentation liquid obtained in Example 1 was used as the test sample to study the anti-myocardial ischemia ability of the American ginseng fermentation liquid in rats.
[0078] Forty male SPF-grade SD rats, 8 weeks old and weighing 180±10g, were used in the experiment. After 7 days of acclimatization, the rats were randomly divided into a blank group, a model group, a fermented American ginseng liquid group, and an unfermented American ginseng extract group, with 10 rats in each group.
[0079] Ischemia models were established in the left anterior descending branch of the heart of rats in the ligation model group, the American ginseng fermentation liquid group, and the unfermented American ginseng extract group.
[0080] Based on the human / rat dosage conversion, fermented American ginseng broth and unfermented American ginseng extract were administered orally at a dose equivalent to 0.27 g / kg of raw material per rat, with 2 mL administered per rat. The fermented American ginseng broth was diluted with distilled water, and 2 mL of distilled water was administered per rat in the model group and control group. One hour after the last administration, the ejection fraction of the rats was collected using an animal ultrasound machine. Subsequently, the rats were anesthetized with isoflurane, sacrificed, and their heart tissue was harvested. The calcium content of the rat heart tissue was measured using a commercially available kit. 2+ -ATPase and Na +- ATPase content. The experimental procedure followed the 3R principle of animal experiments.
[0081] The test results are as follows:
[0082] Compared with unfermented American ginseng extract, fermented American ginseng broth significantly increased the ejection fraction in rats with myocardial ischemia. (See results below.) Figure 3 Further detection of Ca 2+ -ATPase and Na + -ATPase activity, results are shown in Figure 4 The regulatory capacity of fermented American ginseng broth is far stronger than that of unfermented American ginseng extract. In vivo animal studies have confirmed that fermented American ginseng broth has stronger medicinal efficacy and higher application value.
[0083] Example 7
[0084] The ginseng fermentation liquid obtained in Example 1 was used as the test sample to study the vasodilatory ability of the ginseng fermentation liquid.
[0085] Twenty male SPF-grade SD rats, 12 weeks old and weighing 260±10g, were used in the experiment. After 7 days of acclimatization, the rats were randomly divided into a fermented American ginseng liquid group and an unfermented American ginseng extract group, with 10 rats in each group. Baseline blood pressure values were collected using a non-invasive blood pressure measurement system for rats and mice. Subsequently, the control group, fermented American ginseng liquid group, and unfermented American ginseng extract group were administered the same via gavage as in Example 6. After 3 days of oral gavage, blood pressure values were measured using a non-invasive blood pressure measurement system for rats and mice 1 hour after the last administration, and the changes in blood pressure values before and after gavage were compared. The experimental procedure followed the 3R principle of animal experiments.
[0086] The test results are as follows:
[0087] The test results are shown in Figure 5 The experiment used 12-week-old rats, which are older and have a higher vascular risk. Compared with the unfermented American ginseng extract, the fermented American ginseng liquid had a higher downregulation value for diastolic and systolic blood pressure, and had a stronger blood pressure lowering function and a stronger ability to regulate blood pressure.
[0088] Experiments have shown that the American ginseng fermented liquid obtained in Examples 2 and 3, which has anti-myocardial ischemia or vasodilatory effects, has similar oral toxicity tolerance test in mice, anti-myocardial ischemia ability in rats, and vasodilatory ability to the same as the American ginseng fermented liquid obtained in Example 1.
Claims
1. A method for preparing American ginseng fermentation liquid with anti-myocardial ischemia or vasodilatory effects, characterized in that: Includes the following steps: 1) Add purified water to American ginseng, heat and reflux to extract, filter, combine the filtrates, concentrate the filtrate to obtain concentrated solution; 2) Mix the concentrated solution with the OD solution at a volume ratio of 95-99:5-1. 600 Mix with MRS medium containing Lactobacillus plantarum with a pH value in the range of 0.5-0.9, and ferment in a sealed container at 35-39℃ and 10-60 rpm for 24-48 hours to obtain crude fermentation broth; inactivate, centrifuge, and obtain supernatant; The Lactobacillus plantarum is Lactobacillus plantarum ATCC8014; 3) The supernatant is concentrated under reduced pressure at 50-60℃ to 1 / 3 to 2 / 3 of the supernatant volume to obtain American ginseng fermentation liquid.
2. The preparation method according to claim 1, characterized in that: Step 1) is as follows: Add purified water to American ginseng, heat and reflux to extract twice, and the mass of purified water added each time is 8-12 times and 7-9 times the mass of American ginseng, respectively; each extraction lasts 1.5-3 hours, filter, combine the filtrates, and concentrate under reduced pressure at 50-60℃ to a mass equivalent to 4-6 times the mass of American ginseng to obtain a concentrated solution.
3. The preparation method according to claim 1, characterized in that: The inactivation steps are as follows: raise the temperature to 55℃-65℃, maintain for 25-35 minutes, then raise the temperature to 70℃-80℃ and maintain for 3-10 minutes.
4. The preparation method according to claim 1, characterized in that: The centrifugation speed is 2000-4000 rpm, and the centrifugation time is 10-20 min.
5. A fermented American ginseng liquid with anti-myocardial ischemia or vasodilatory effects prepared by the preparation method of any one of claims 1-4.
6. The use of the American ginseng fermentation liquid according to claim 5 in the preparation of anti-myocardial ischemia products.
7. The use of the American ginseng fermentation liquid according to claim 5 in the preparation of vasodilator products.