Rare ginsenoside composition and its role in anti-fatigue and liver protection

Abstract

The present application discloses a rare ginsenoside composition and its application in anti-fatigue and liver protection, and relates to the technical field of biological medicine. The present application provides a rare ginsenoside composition, which is composed of Rh4, F4 and Dehydroprotopanaxatrial II, and the mass ratio of Rh4, F4 and Dehydroprotopanaxatrial II is (2-4):(2-4):1. The rare ginsenoside composition has the effect of significantly relieving physical fatigue, can reduce the decomposition and metabolism of protein to energy, eliminate fatigue, increase the liver glycogen reserve in the body, improve the fatigue resistance, has the effect of liver protection, enhances the antioxidant defense ability of liver tissue, reduces the lipid peroxidation damage, and improves the lipid metabolism disorder of liver tissue.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to rare ginsenoside compositions and their application in anti-fatigue and liver protection. Background Technology

[0002] Ginseng, a perennial herb belonging to the genus Panax in the family Araliaceae, has core effects in nourishing vital energy, strengthening the spleen and lungs, promoting body fluid production and nourishing blood, calming the mind and improving intelligence. It is widely used to improve symptoms such as physical weakness, cold limbs and weak pulse, spleen deficiency and poor appetite, and lung deficiency and cough.

[0003] Ginsenosides are the core material basis for the pharmacological effects of ginseng. Based on structural differences, ginsenosides can be divided into common ginsenosides (such as Rg1, Rb1, Rc, etc.) and rare ginsenosides (such as Rh2, Rg3, Rh4, F4, etc.). Rare ginsenosides, due to their extremely low natural content in fresh or dried ginseng, require processes such as enzymatic hydrolysis, microbial transformation, and high-temperature, high-pressure extraction to be obtained from or enriched from common ginsenosides. However, their biological activity is far higher than that of common ginsenosides. Studies have shown that rare ginsenosides can more easily penetrate cell membranes and the blood-brain barrier, exhibiting more significant pharmacological effects in anti-fatigue, anti-inflammatory, hepatoprotective, and immunomodulatory effects. Therefore, they have become a core focus of ginseng pharmacological research in recent years.

[0004] Chronic fatigue syndrome and physiological fatigue have become widespread health problems. From a physiological perspective, fatigue is closely related to the body's energy metabolism disorder: when the body is under high-intensity activity or long-term stress, the body's glycogen reserves are rapidly depleted. If the energy supply is insufficient, the body will initiate protein catabolism to replenish energy, leading to muscle protein degradation and increased fatigue. At the same time, the oxidative stress response accompanying fatigue will further aggravate cell damage.

[0005] Currently, anti-fatigue products on the market are mainly divided into two categories: one is chemically synthesized products that rely on stimulating ingredients such as caffeine and taurine. Although they can refresh the mind in the short term, long-term use can easily lead to side effects such as insomnia and palpitations, and they cannot fundamentally improve energy metabolism disorders; the other is products based on natural ingredients, among which products with common ginsenosides as the core ingredient are the most common. However, due to the low bioavailability of common ginsenosides, their effect on relieving physical fatigue is limited.

[0006] As the largest metabolic organ in the human body, the liver undertakes core physiological functions such as detoxification, lipid metabolism, and antioxidant defense. However, it is susceptible to damage from external factors (such as long-term alcohol consumption, drug abuse, high-fat diets, and staying up late), leading to pathological changes such as lipid peroxidation, decreased antioxidant capacity, and lipid metabolism disorders, which can eventually develop into fatty liver, liver fibrosis, and even cirrhosis. From a pathological mechanism perspective, the core link in liver damage lies in the excessive generation of reactive oxygen species (ROS). ROS attack the lipid components on the liver cell membrane, triggering lipid peroxidation and causing damage to the cell membrane structure. At the same time, ROS also inhibits the activity of antioxidant enzymes such as glutathione and superoxide dismutase, weakening the liver tissue's antioxidant defense system and further aggravating liver cell damage.

[0007] Liver protection products mainly include chemical drugs (such as polyene phosphatidylcholine and silymarin) and natural extracts (such as wolfberry polysaccharides and Ganoderma lucidum triterpenes). While chemical drugs can improve liver damage indicators in the short term, long-term use may lead to side effects such as gastrointestinal discomfort and drug-induced liver injury. Natural extract products, on the other hand, generally suffer from unclear active ingredients and ambiguous mechanisms of action.

[0008] Therefore, developing a rare ginsenoside composition consisting of Rh4, F4, and Dehydroprotopanaxatrial II in a specific ratio to simultaneously relieve physical fatigue and protect the liver, thus meeting the market's urgent need for safe, efficient, and multifunctional natural active compositions, has significant academic value and application prospects. Summary of the Invention

[0009] The purpose of this invention is to provide rare ginsenoside compositions and their application in the preparation of drugs for relieving physical fatigue and protecting the liver.

[0010] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows:

[0011] In a first aspect, the present invention provides a rare ginsenoside composition, wherein the rare ginsenoside composition is composed of Rh4, F4 and Dehydroprotopanaxatrial II, and the mass ratio of Rh4, F4 and Dehydroprotopanaxatrial II is (2-4):(2-4):1.

[0012] According to some embodiments of the present invention, the mass ratio of Rh4, F4 and Dehydroprotopanaxatrial II in the rare ginsenoside composition can be 2:2:1, 3:3:1, 4:4:1, 2:3:1, 2:4:1, 3:2:1, 3:4:1, 4:2:1, 4:3:1, and any intermediate value can be selected.

[0013] Specifically, the mass ratio of Rh4, F4 and Dehydroprotopanaxatrial II is 2:2:1, 3:3:1 or 4:4:1.

[0014] Furthermore, the mass ratio of Rh4, F4, and Dehydroprotopanaxatrial II is 3:3:1.

[0015] Secondly, the present invention provides a method for preparing the above-mentioned rare ginsenoside composition, comprising the following steps:

[0016] (1) Take the total saponin powder of ginseng stems and leaves, add anhydrous ethanol, concentrate under reduced pressure to obtain preliminary ginseng extract;

[0017] (2) Dissolve the preliminary ginseng extract in step (1), use ether and n-butanol as solvents in sequence, shake to treat, collect the supernatant, concentrate to obtain the secondary refined extract;

[0018] (3) After extraction, β-glucanase was added to the reaction system, and the mixture was placed in a shaker to maintain the temperature at 40℃-50℃ and the pH at 6.5-7.5 for the reaction. After the reaction was completed, the mixture was centrifuged to prepare the preliminary product.

[0019] (4) The preliminary product was separated and purified by C-18 semi-preparative high performance liquid chromatography to obtain high purity Rh4, F4 and Dehydroprotopanaxatrial II monomers, which were then mixed according to the mass ratio to obtain a rare ginsenoside composition.

[0020] According to some embodiments of the present invention, the amount of anhydrous ethanol added in step (1) is 8-12 times the volume;

[0021] Furthermore, the amount of anhydrous ethanol added in step (1) is 10 times the volume;

[0022] According to some embodiments of the present invention, the conditions for vacuum concentration in step (1) are 50-60°C and 0.05-0.07 MPa;

[0023] Furthermore, the conditions for vacuum concentration in step (1) are 60°C and 0.06 MPa.

[0024] According to some embodiments of the present invention, the amount of diethyl ether and n-butanol added in step (2) is 1-3 times the volume;

[0025] Furthermore, in step (2), the amount of diethyl ether and n-butanol added is 1 volume;

[0026] According to some embodiments of the present invention, the conditions for the oscillation treatment in step (2) are: treatment for 2 hours at 100°C and with an oscillation and vibration power of 600 W respectively.

[0027] According to some embodiments of the present invention, the amount of β-glucanase added in step (3) is one-thousandth to ten-thousandths;

[0028] Furthermore, the amount of β-glucanase added in step (3) is 0.5%.

[0029] According to some embodiments of the present invention, the reaction time in step (3) is 1-3 hours;

[0030] Furthermore, the reaction time in step (3) is 2 hours.

[0031] Thirdly, the present invention provides the application of the above-mentioned rare ginsenoside composition in the preparation of anti-fatigue products.

[0032] Specifically, the products mentioned include health supplements and medicines.

[0033] Furthermore, when the product is a drug, the drug has at least one of the following effects:

[0034] (1) Anti-fatigue;

[0035] (2) Reduce serum urea nitrogen content and decrease protein breakdown for energy;

[0036] (3) Increase liver glycogen levels and improve fatigue resistance.

[0037] Specifically, when the product is a drug, the drug includes a pharmaceutically acceptable carrier.

[0038] Furthermore, the pharmaceutically acceptable carrier is selected from one or more excipients, stabilizers, diluents, binders, preservatives, and lubricants.

[0039] Specifically, the dosage form of the drug is drops, mixture, tincture, injection, tablet, powder, oral liquid, capsule, granule, ointment, suspension, powder, emulsion, solution, drop pill, pill, lozenge, lyophilized powder for injection, gel, suppository or aerosol.

[0040] Furthermore, the health food also includes conventional excipients for health foods, including but not limited to fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional fortifiers.

[0041] Furthermore, the health food products mentioned include emulsion products, solution products, powder products, and solid products.

[0042] Specifically, the product shown contains a rare ginsenoside composition as the main active ingredient.

[0043] Fourthly, the present invention provides the use of the above-mentioned rare ginsenoside composition in the preparation of products for relieving or treating alcoholic liver injury.

[0044] Furthermore, the alcoholic liver injury mentioned above includes acute alcoholic liver injury and chronic alcoholic liver injury.

[0045] Specifically, the product in question is a medicine or health supplement.

[0046] Specifically, when the product is a drug, the drug has at least one of the following effects:

[0047] (1) Reduce serum ALT and AST levels;

[0048] (2) Increase the levels of GSH and SOD in liver tissue;

[0049] (3) Reduces MDA levels in liver tissue;

[0050] (4) Reduces TG levels in liver tissue;

[0051] (5) Improves the degree of inflammatory infiltration of hepatocytes;

[0052] (6) Reduce serum ROS levels;

[0053] (7) Increase serum GSH-PX levels;

[0054] (8) Reduce serum IL-6 levels.

[0055] Specifically, when the product is a drug, the drug includes a pharmaceutically acceptable carrier.

[0056] Furthermore, the pharmaceutically acceptable carrier is selected from one or more excipients, stabilizers, diluents, binders, preservatives, and lubricants.

[0057] Specifically, the dosage form of the drug is drops, mixture, tincture, injection, tablet, powder, oral liquid, capsule, granule, ointment, suspension, powder, emulsion, solution, drop pill, pill, lozenge, lyophilized powder for injection, gel, suppository or aerosol.

[0058] Specifically, the product contains rare ginsenoside composition as the main active substance.

[0059] The beneficial effects of this invention are as follows:

[0060] This invention provides a rare ginsenoside composition composed of Rh4, F4, and Dehydroprotopanaxatrial II. This rare ginsenoside composition significantly alleviates physical fatigue, reduces protein breakdown for energy, eliminates fatigue, increases liver glycogen reserves, and improves fatigue resistance. The rare ginsenoside composition also has a hepatoprotective effect, enhancing the antioxidant defense capacity of liver tissue, reducing lipid peroxidation damage, and improving lipid metabolism disorders in liver tissue. Attached Figure Description

[0061] Figure 1 The diagram shows the preparation chromatograms of ginsenoside F4, ginsenoside Rh4, and Dehydroprotopanaxatrial II in Experimental Example 1.

[0062] Figure 2 The chromatographic peaks are those of ginsenoside F4 standard (A) and the prepared F4 sample (B).

[0063] Figure 3 Chromatographic peaks of Rh4 standard (A) and prepared Rh4 sample (B).

[0064] Figure 4 Chromatographic peaks of Dehydroprotopanaxatrial II standard (A) and the prepared Dehydroprotopanaxatrial II sample (B).

[0065] Figure 5 The time to exhaustion during weight-bearing swimming for each group of mice was (n ± SEM, n=6). p <0.001, ▲▲ p <0.01, ▲▲▲ p <0.001.

[0066] Figure 6 The effect of each group on serum urea nitrogen in mice (n ± SEM, n=6). p <0.05, p <0.01, ▲ p <0.05, ▲▲ p <0.01.

[0067] Figure 7 The effect of each group on liver glycogen in mice (n ± SEM, n=6). p <0.001, ▲▲ p <0.01, ▲▲▲ p <0.001.

[0068] Figure 8 Serum ALT(A) and AST(B) levels (n ± SEM, n=6). ### p <0.001, p <0.01, p <0.001, ▲ p <0.05, ▲▲ p <0.01, ▲▲▲ p <0.001.

[0069] Figure 9 The levels of GSH(A), SOD(B), and MDA(C) in liver tissue (n ± SEM, n=6) # p <0.05, ### p <0.001, p <0.05, p <0.01, ▲ p <0.05, ▲▲ p <0.01, ▲▲▲ p <0.001.

[0070] Figure 10 The TG level in liver tissue is (n ± SEM, n=6). ### p <0.001, p <0.01, ▲▲ p <0.01.

[0071] Figure 11 Image of H&E staining on liver slices.

[0072] Figure 12 Serum ALT(A) and AST(B) levels (n ± SEM, n=6). ### p <0.001, p <0.01, p <0.001, ▲ p <0.05, ▲▲ p <0.01.

[0073] Figure 13 The levels of MDA (A) and SOD (B) in serum (n ± SEM, n=6) # p <0.05, ### p <0.001, p <0.05, p <0.01, p <0.001, ▲▲ p <0.01, ▲▲▲ p <0.001.

[0074] Figure 14 The levels of ROS (A) and GSH-PX (B) in serum (n ± SEM, n=6) ## p <0.01, ### p <0.001, p <0.05, p <0.001, ▲ p <0.05, ▲▲ p <0.01.

[0075] Figure 15 Serum IL-6 levels (n ± SEM, n=6). ### p<0.001, p <0.01, ▲ p <0.05, ▲▲ p <0.01. Detailed Implementation

[0076] To make the technical means, creative features, and achieved objectives and effects of this invention easier to understand, the invention is further illustrated below with specific embodiments. However, the following embodiments are only preferred embodiments of this invention and not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the protection scope of this invention. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used is conventional equipment, and the equipment and materials used in each embodiment are the same.

[0077] In this invention, the CAS number of Dehydroprotopanaxatrial II is 174688-80-3.

[0078] Experiment Example 1: Relieving Physical Fatigue

[0079] 1 Experimental Methods

[0080] 1.1 Experimental Materials

[0081] The components of the rare ginsenoside composition drug are:

[0082] Rh4, F4 and Dehydroprotopanaxatrial II.

[0083] The preparation method of the rare ginsenoside composition is as follows:

[0084] (1) Take the purchased ginseng stem and leaf total saponin powder, add 10 times the volume of anhydrous ethanol, and concentrate it under reduced pressure at 60℃ and 0.06Mpa to obtain preliminary ginseng extract;

[0085] (2) The extract in step (1) is redissolved and placed in an ultrasonic instrument. One volume of diethyl ether and one volume of n-butanol are used as solvents in sequence. The mixture is treated for 2 hours at 100°C with oscillation and vibration power of 600 W. The supernatant after each treatment is collected, combined and concentrated by rotary evaporation to obtain the secondary purified extract.

[0086] (3) Dissolve the extract obtained in step (2) into the reaction system, add 0.5% β-glucanase, and place the mixture in a shaker. Maintain the temperature at 40℃-50℃ and the pH at 6.5-7.5 for 2 hours to complete the preliminary preparation of the product.

[0087] (5) The preliminary product was separated and purified using C-18 semi-preparative high-performance liquid chromatography under the following chromatographic conditions:

[0088] Chromatographic column: C18 column, 300 mm × 7.8 mm, 7 μm;

[0089] Column temperature: 30℃;

[0090] Mobile phase A is 10% acetonitrile, and mobile phase B is 80% acetonitrile;

[0091] Gradient elution conditions: 0-26 min: 57% → 15% A; 26-43 min: 15% → 0% A;

[0092] Flow rate: 3 mL / min;

[0093] The detection wavelength is 203 nm;

[0094] Preparation chromatograms are shown below Figure 1 Ginsenoside F4 was collected at 8-10 min, ginsenoside Rh4 at 11-13 min, and Dehydroprotopanaxatrial II at 29-31 min.

[0095] The collected compounds were fed into an analytical column under the following chromatographic conditions:

[0096] Chromatographic column: Waters C18 column, 4.6 mm × 250 mm, 5 μm;

[0097] Column temperature: 30℃;

[0098] Mobile phase A is 10% acetonitrile, and mobile phase B is 80% acetonitrile;

[0099] The gradient elution conditions were as follows: 0-14 min, 86%→84% A; 14-24 min, 84%→83% A; 24-36 min, 83%→81% A; 36-60 min, 81%→57% A; 60-82 min, 57%→15% A; 82-98 min, 15%→0% A; 98-110 min, 0%→100% A.

[0100] Flow rate: 0-60 min: 1.3 mL / min, 60-109.5 min: 1.0 mL / min;

[0101] Detection wavelength: 203 nm;

[0102] Among them, the peak elution time of ginsenoside F4 was 68.722 min, the peak elution time of ginsenoside Rh4 was 70.285 min, and the peak elution time of Dehydroprotopanaxatrial II was 87.552 min.

[0103] Figure 2 In the chromatographic peak, A represents ginsenoside F4 standard (Chengdu Mansite Biotechnology Co., Ltd.). Figure 2 B in the figure represents the chromatographic peak of the prepared F4 sample.

[0104] Figure 3 In the figure, A represents the chromatographic peak of Rh4 standard (Chengdu Mansite Biotechnology Co., Ltd.). Figure 3 B in the figure represents the chromatographic peak of the prepared Rh4 sample.

[0105] Figure 4 In the figure, A represents the chromatographic peak of Dehydroprotopanaxatrial II standard (Shanghai Yuanye Biotechnology Co., Ltd.). Figure 4 B in the figure represents the chromatographic peak of the prepared Dehydroprotopanaxatrial II sample.

[0106] The obtained high-purity Rh4, F4 and Dehydroprotopanaxatrial II monomers were mixed in a ratio of 3:3:1 to obtain a rare ginsenoside composition.

[0107] Blood urea nitrogen (BUN) ELISA kit (Hangzhou Lianke Biotechnology Co., Ltd.), Glycogen ELISA kit (Hangzhou Lianke Biotechnology Co., Ltd.).

[0108] 1.2 Laboratory Animals

[0109] After one week of acclimatization, C57BL / 6J mice were randomly divided into 8 groups (n=6) according to body weight, including:

[0110] (1) Blank group;

[0111] (2) A rare ginsenoside composition group 1 (80 mg / kg), wherein the mass ratio of Rh4, F4 and Dehydroprotopanaxatrial II is 4:4:1;

[0112] (3) Two groups of rare ginsenoside compositions (80 mg / kg), with the mass ratio of Rh4, F4 and Dehydroprotopanaxatrial II being 2:2:1;

[0113] (4) Three groups of rare ginsenoside compositions (80 mg / kg), with the mass ratio of Rh4, F4 and Dehydroprotopanaxatrial II being 3:3:1;

[0114] (5) Comparative Example 1 (80 mg / kg): The mass ratio of Rh4, F4 and Dehydroprotopanaxatrial II was 0.5:0.5:1;

[0115] (6) Comparative Example 2 (80 mg / kg): The mass ratio of Rh4, F4 and Dehydroprotopanaxatrial II was 5:5:1;

[0116] (7) Comparative Example 3 (80 mg / kg): The mass ratio of Rh4, Rk1 and Dehydroprotopanaxatrial II was 3:3:1;

[0117] (8) Comparative Example 4 (80mg / kg): The mass ratio of Rh4, F4 and Rk3 was 3:3:1.

[0118] The mice were administered 10 ml / kg of the drug via gavage once daily, while the control group received 10 ml / kg of physiological saline once daily for 30 days. Mice were weighed weekly, and the dosage of the test sample was adjusted according to body weight.

[0119] 1.3 Mouse weight-bearing swimming experiment

[0120] The first batch of mice underwent a weight-bearing swimming experiment. Thirty minutes after the last administration of the test sample, the mice were weighed, and a lead weight equal to 5% of their body weight was placed on their tail base before they were placed in a swimming tank (58×43×34 cm) to swim. The water depth was at least 30 cm, and the water temperature was 25 ± 1.0℃. The time from the start of swimming to exhaustion was recorded as the weight-bearing swimming time. If the exhaustion time of the test sample group was significantly longer than that of the control group, and the difference was statistically significant, the experimental result was considered positive.

[0121] 1.4 Establishment of a high urea model

[0122] The second batch of mice was used to establish a high urea model. Thirty minutes after the last administration of the test sample, the mice swam in water at 30°C without load for 90 minutes. After a 60-minute rest, approximately 0.5 ml of whole blood was collected from the eyeballs. After standing for 30 minutes, the blood was centrifuged at 4000 r / min for 15 minutes, and the supernatant serum was collected and stored at -80°C for later use. The urea level in the serum was detected using an ELISA assay according to the kit instructions (manufacturer: Hangzhou Lianke Biotechnology Co., Ltd.).

[0123] 1.5 Determination of liver glycogen

[0124] The third batch of mice was used for liver glycogen level determination. Animals were sacrificed 30 minutes after the last sample administration, and the left lobe of the liver was harvested. Liver glycogen levels in the liver homogenate were measured using ELISA according to the kit instructions (manufacturer: Hangzhou Lianke Biotechnology Co., Ltd.).

[0125] 1.6 Data Statistics

[0126] Data are expressed as mean ± standard error (Mean ± SEM). Statistical analysis was performed using GraphPadPrism 10.1.2 software. First, one-way ANOVA was used to assess the overall differences between groups. If significant differences were found, Tukey's multiple comparison method was used to perform pairwise comparisons between groups to clarify the sources of difference. The statistical significance level was set at [value missing]. p <0.05.

[0127] 2. Experimental Results

[0128] 2.1 Effects of rare ginsenoside combination drugs on weight-bearing swimming time in mice

[0129] The time to exhaustion in the weighted swimming test is the most direct indicator of the body's fatigue resistance. The length of the time to exhaustion in weighted swimming directly reflects the strength of the mouse's fatigue resistance. (From Table 1 and...) Figure 5 It can be seen that, compared with the blank group, the rare ginsenoside composition drug group 1 ( p <0.001), two groups of rare ginsenoside compositions ( p <0.001), three groups of rare ginsenoside compositions ( p <0.001) The time to exhaustion during weight-bearing swimming was significantly increased, and rare ginsenoside composition 1-rare ginsenoside composition 3 were significantly more effective than all comparative groups in relieving physical fatigue. p <0.001, p <0.001, p <0.001, p<0.05). The results showed that the rare ginsenoside composition drug could significantly alleviate physical fatigue in mice.

[0130] Table 1. Time to exhaustion during weight-bearing swimming in each group of mice (n ± SEM, n=6)

[0131]

[0132] Note: Compared to the blank group, p <0.001; compared to rare ginsenoside composition 3, ▲▲ p <0.01, ▲▲▲ p <0.001.

[0133] 2.2 Effects of rare ginsenoside combination drugs on serum urea in mice

[0134] Serum urea nitrogen levels in animals are directly proportional to serum urea levels. Blood urea nitrogen is a metabolic product of proteins and amino acids in animals; elevated blood urea nitrogen levels can induce exercise-induced fatigue. Therefore, blood urea nitrogen is one of the important indicators for evaluating the body's anti-fatigue ability. (From Table 2 and...) Figure 6 It can be seen that, compared with the blank group, the rare ginsenoside composition group 1 ( p <0.05), two groups of rare ginsenoside compositions ( p <0.05), three groups of rare ginsenoside compositions ( p <0.01) Serum urea nitrogen levels were significantly reduced, and rare ginsenoside composition 1-rare ginsenoside composition 3 were significantly more effective than all comparative groups in reducing serum urea nitrogen levels. p <0.05, p <0.01, p <0.05, p <0.01). The results showed that the rare ginsenoside composition could reduce the serum urea nitrogen content in mice after swimming, indicating that the rare ginsenoside composition can reduce the catabolism of protein for energy and eliminate fatigue.

[0135] Table 2. Serum urea nitrogen levels in mice of each group (n ± SEM, n=6)

[0136]

[0137] Note: Compared to the blank group, p <0.05, p<0.01; compared to rare ginsenoside composition 3, ▲ p <0.05, ▲▲ p <0.01.

[0138] 2.3 Effects of rare ginsenoside compositions on mouse liver glycogen

[0139] Glycogen is the body's primary energy source and plays a crucial role in maintaining blood glucose levels during prolonged or high-intensity exercise. Related studies have shown that the relative stability of blood glucose levels is mainly maintained by liver glycogen; its depletion leads to hypoglycemia, which in turn causes decreased exercise endurance and damage to the nervous system. Therefore, liver glycogen reserve levels can serve as an important indicator of the body's fatigue resistance. (See Table 3 and...) Figure 7 It can be seen that compared with the blank group, the rare ginsenoside composition group 1 ( p <0.001), two groups of rare ginsenoside compositions ( p <0.001), three groups of rare ginsenoside compositions ( p <0.001) significantly increased liver glycogen reserves, and rare ginsenoside composition 1-rare ginsenoside composition 3 were significantly more effective than all comparative groups in reducing serum urea nitrogen levels. p <0.001, p <0.001, p <0.001, p <0.01). The results showed that the rare ginsenoside composition could significantly increase liver glycogen reserves in mice and greatly improve their fatigue resistance.

[0140] Table 3. Liver glycogen levels in mice of each group (n ± SEM, n=6)

[0141]

[0142] Note: Compared to the blank group, p <0.001; compared to rare ginsenoside composition 3, ▲▲ p <0.01, ▲▲▲ p <0.001.

[0143] Experimental Example 2: Liver Protective Effect

[0144] 1. Alleviate alcoholic liver damage

[0145] 1.1 Experimental Methods

[0146] 1.1.1 Experimental Materials

[0147] The experimental reagents are shown in Table 4.

[0148] Table 4 Experimental Reagents

[0149]

[0150] 1.1.2 Laboratory Animals

[0151] Adult SPF-grade male Kunming mice were randomly divided into 10 groups:

[0152] (1) Blank group;

[0153] (2) Model group;

[0154] (3) Positive control group: Biphenyl diester droplets (7.5 mg / kg);

[0155] (4) A group of rare ginsenoside composition drugs (80 mg / kg), with the mass ratio of Rh4, F4 and Dehydroprotopanaxatrial II being 4:4:1;

[0156] (5) Two groups of rare ginsenoside compositions (80 mg / kg), with the mass ratio of Rh4, F4 and Dehydroprotopanaxatrial II being 2:2:1;

[0157] (6) Three groups of rare ginsenoside compositions (80 mg / kg), with the mass ratio of Rh4, F4 and Dehydroprotopanaxatrial II being 3:3:1;

[0158] (7) Comparative Example 1 (80 mg / kg): The mass ratio of Rh4, F4 and Dehydroprotopanaxatrial II was 0.5:0.5:1;

[0159] (8) Comparative Example 2 (80 mg / kg): The mass ratio of Rh4, F4 and Dehydroprotopanaxatrial II was 5:5:1;

[0160] (9) Comparative Example 3 (80 mg / kg): The mass ratio of Rh4, Rk1 and Dehydroprotopanaxatrial II was 3:3:1;

[0161] (10) Comparative Example 4 (80mg / kg): The mass ratio of Rh4, F4 and Rk3 was 3:3:1.

[0162] The mice were administered 10 ml / kg once daily by gavage for 30 days. The mice were weighed weekly, and the dosage of the test sample was adjusted according to body weight.

[0163] 1.1.3 Modeling Method

[0164] At the end of the administration of the test samples, the model group and each drug administration group were given 50% ethanol (12 mL / kg) by gavage once, while the blank control group was given distilled water. After fasting for 16 hours, blood was collected and the animals were euthanized.

[0165] 1.1.4 Sample Preparation

[0166] Serum samples: After modeling, mice were fasted for 16 hours. Blood was collected from the eyes of each mouse (approximately 0.5 ml per mouse). After standing for 30 min, the blood was centrifuged at 4000 r / min for 15 min. The supernatant serum was collected and stored at -80℃ for later use. AST and ALT activities in the serum were detected according to the kit instructions. Liver samples: Mice were sacrificed after collecting blood from the eyes. The liver was harvested. Part of the liver was fixed with 4% polyformaldehyde, and the remaining liver was homogenized for the determination of MDA, GSH, SOD, TG, and other indicators.

[0167] Histopathological examination: After the mice were euthanized, the livers were removed and cross-sections were taken from the middle of the left lobe. The liver tissue was fixed with 4% paraformaldehyde for 48 hours, then embedded in paraffin, sectioned, and stained with H&E. The characteristics of the liver tissue were observed under a microscope.

[0168] 1.1.5 Data Statistics

[0169] Data are expressed as mean ± standard error (Mean ± SEM). Statistical analysis was performed using GraphPadPrism 10.1.2 software. First, one-way ANOVA was used to assess the overall differences between groups. If significant differences were found, Tukey's multiple comparison method was used to perform pairwise comparisons between groups to clarify the sources of difference. The statistical significance level was set at [value missing]. p <0.05.

[0170] 1.2 Experimental Results

[0171] 1.2.1 Effects on serum ALT and AST levels

[0172] ALT and AST are key enzymes involved in amino acid metabolism within hepatocytes, functioning within the cells. Under normal circumstances, their levels in the blood are low. In alcoholic liver injury, cell membrane permeability increases, leading to elevated ALT and AST levels, especially AST, which is a sensitive marker of acute alcoholic liver injury. Serum ALT and AST levels are shown in Table 5. Figure 8 As shown. Compared with the blank group, the serum ALT and AST levels in the model group mice were significantly increased ( p<0.001 indicates that the ALD model was successfully established. Compared with the model group, the serum ALT and AST levels in the positive drug biphenyl diester droplet group were both decreased ( p <0.01, p <0.01); the rare ginsenoside composition drug group 1, rare ginsenoside composition drug group 2, and rare ginsenoside composition drug group 3 significantly reduced ALT levels ( p <0.001, p <0.01, p <0.01, significantly reduced AST level ( p <0.001, p <0.001, p <0.01%, rare ginsenoside composition 1-rare ginsenoside composition 3 showed significantly better effects on reducing ALT levels than all comparative groups ( p <0.01, p <0.01, p <0.01, p <0.001), rare ginsenoside composition 1-rare ginsenoside composition 3 showed significantly better effects on reducing AST levels than all comparative groups. p <0.05, p <0.01, p <0.05, p <0.01).

[0173] Table 5. ALT and AST levels in mice of each group (n ± SEM, n=6)

[0174]

[0175] Note: Compared to the blank group, ### p <0.001; compared to the model group, p <0.01, p <0.001; compared to rare ginsenoside composition 3, ▲ p <0.05, ▲▲ p <0.01, ▲▲▲ p <0.001.

[0176] 1.2.2 Effects on the levels of GSH, MDA, and SOD in liver homogenate

[0177] Alcoholic liver injury generates a large number of free radicals, triggering oxidative stress and damaging hepatocytes. GSH and SOD, as important antioxidants, play a crucial role in resisting oxidative damage, and their levels reflect the body's antioxidant capacity. (See Table 6 and...) Figure 9 As shown, compared with the blank group, the levels of GSH and SOD in the model group mice were significantly reduced ( p <0.001, p <0.05%, the level of MDA in the oxidation product was significantly increased ( p <0.001, indicating that alcoholic liver injury leads to decreased antioxidant capacity, increased oxidative stress, and aggravated lipid peroxidation in mouse liver tissue, thus causing oxidative damage to liver tissue. Compared with the model group, gavage administration of biphenyl diester drops significantly increased GSH and SOD levels in the liver tissue of mice with liver injury ( p <0.01, p <0.05), and simultaneously, gavage administration of rare ginsenoside composition group 1, rare ginsenoside composition group 2, and rare ginsenoside composition group 3 also significantly increased the levels of GSH and SOD in liver tissue ( p <0.01, p <0.001). Rare ginsenoside composition 1-Rare ginsenoside composition 3 showed significantly better effects on increasing GSH levels than all comparative groups. p <0.01, p <0.01, p <0.01, p <0.05), rare ginsenoside composition 1-rare ginsenoside composition 3 showed significantly better effects on increasing SOD levels than all comparative groups. p <0.01, p <0.01, p <0.01, p The result of <0.01 indicates that rare ginsenoside composition 1, rare ginsenoside composition 2, and rare ginsenoside composition 3 have significant protective effects against alcoholic liver injury and can enhance the antioxidant defense capacity of liver tissue.

[0178] Malondialdehyde (MDA) is the end product of lipid peroxidation, and its level reflects the degree of intracellular lipid peroxidation. The model group showed a significantly elevated MDA level. p <0.001 indicates that severe lipid peroxidation occurred in liver tissue during alcoholic liver injury. This is because a large number of free radicals produced by alcohol metabolism attack polyunsaturated fatty acids on cell membranes, triggering a chain reaction of lipid peroxidation and leading to increased MDA production. Compared with the model group, the MDA level in the positive control group (biphenyl diester drops) was significantly reduced ( p<0.001), and simultaneously, rare ginsenoside composition group 1, rare ginsenoside composition group 2, and rare ginsenoside composition group 3 also significantly reduced the MDA level in liver tissue ( p <0.01, p <0.01, p <0.01), and rare ginsenoside composition 1-rare ginsenoside composition 3 were significantly more effective than all comparative groups in increasing MDA levels. p <0.001, p <0.01, p <0.01, p The value <0.01 indicates that the rare ginsenoside composition has the effect of reducing MDA levels and alleviating lipid peroxidation damage.

[0179] Table 6. GSH, SOD and MDA levels in mice of each group (n ± SEM, n=6)

[0180]

[0181] Note: Compared to the blank group, # p <0.05, ### p <0.001; compared to the model group, p <0.05, p <0.01; compared to rare ginsenoside composition 3, ▲ p <0.05, ▲▲ p <0.01, ▲▲▲ p <0.001.

[0182] 1.2.3 Effect on TG levels in liver homogenate

[0183] Excessive accumulation of triglycerides in liver tissue is a key pathological feature of alcoholic liver injury. Excess triglycerides accumulate in hepatocytes as lipid droplets, leading to hepatic steatosis. As the degree of steatosis worsens, hepatocytes swell and rupture, subsequently triggering inflammatory responses and hepatocyte necrosis. (See Table 7 and...) Figure 10 As shown, compared with the blank group, the level of triglycerides (TG) in the liver tissue of mice in the model group was significantly increased ( p <0.001). Compared with the model group, gavage administration of biphenyl diester drops significantly reduced TG levels in liver tissue of liver-injured mice ( p<0.01), while rare ginsenoside composition drug 1, rare ginsenoside composition drug 2, and rare ginsenoside composition drug 3 have similar effects to biphenyl diester drops ( p <0.01, p <0.01, p <0.01, p <0.01), and rare ginsenoside composition 1-rare ginsenoside composition 3 were significantly more effective than all comparative groups in reducing TG levels. p <0.01, p <0.01, p <0.01, p The result was <0.01, indicating that the rare ginsenoside composition drug can improve lipid metabolism disorder in the liver tissue of mice with alcoholic liver injury, reduce TG levels, alleviate hepatic steatosis, and play a protective role in the liver.

[0184] Table 7. TG levels in mice of each group (n ± SEM, n=6)

[0185]

[0186] Note: Compared to the blank group, ### p <0.001; compared to the model group, p <0.01; compared to rare ginsenoside composition 3, ▲▲ p <0.01.

[0187] 1.2.4 Liver pathological and histological examination

[0188] The H&E staining results of liver sections from mice in each group are as follows: Figure 11The results showed that in the control group, mouse hepatocytes were round and plump, with intact lobular structures, clear and regularly arranged lamina, abundant eosinophilic cytoplasm, no obvious dilation or compression of sinusoids, and no obvious inflammatory cell infiltration. In the model group, hepatocytes showed widespread inflammatory infiltration, large necrotic foci, occasional punctate necrosis, fragmented nuclei, intact lobular structures, blurred lamina structures, significant hepatocyte edema, diffuse vesicles, and diffuse fatty degeneration. Fatty degeneration is the earliest and most common histological manifestation of alcoholic liver injury, thus confirming the successful establishment of the ALD mouse model. In the positive control group (biphenyl diester), the lobular structure was basically intact, and the lamina were neatly arranged. Although a few hepatocytes showed necrosis, the overall condition was mild, with a small amount of inflammatory cell infiltration. In the high, medium, and low dose groups, the rare ginsenoside composition effectively improved the degree of inflammatory infiltration of hepatocytes, making the hepatocyte and lobular structures more intact, the liver plates arranged regularly and neatly, with a few necrotic foci visible, no obvious inflammatory cell infiltration, and a significant reduction in the degree of fatty degeneration. This suggests that the rare ginsenoside composition has a dose-dependent hepatoprotective effect.

[0189] 2. Anti-acute liver injury

[0190] 2.1 Experimental Methods

[0191] 2.1.1 Experimental Materials

[0192] Rare ginsenoside combination drugs.

[0193] The experimental reagents and kits are shown in Table 8.

[0194] Table 8 Experimental drugs, reagents and kits

[0195]

[0196] 2.1.2 Laboratory Animals

[0197] Adult SPF-grade male Kunming mice were randomly divided into 10 groups:

[0198] (1) Blank group;

[0199] (2) Model group;

[0200] (3) Positive control group: Biphenyl diester droplets (7.5 mg / kg);

[0201] (4) A group of rare ginsenoside composition drugs (80 mg / kg), with the mass ratio of Rh4, F4 and Dehydroprotopanaxatrial II being 4:4:1;

[0202] (5) Two groups of rare ginsenoside compositions (80 mg / kg), with the mass ratio of Rh4, F4 and Dehydroprotopanaxatrial II being 2:2:1;

[0203] (6) Three groups of rare ginsenoside compositions (80 mg / kg), with the mass ratio of Rh4, F4 and Dehydroprotopanaxatrial II being 3:3:1;

[0204] (7) Comparative Example 1 (80 mg / kg): The mass ratio of Rh4, F4 and Dehydroprotopanaxatrial II was 0.5:0.5:1;

[0205] (8) Comparative Example 2 (80 mg / kg): The mass ratio of Rh4, F4 and Dehydroprotopanaxatrial II was 5:5:1;

[0206] (9) Comparative Example 3 (80 mg / kg): The mass ratio of Rh4, Rk1 and Dehydroprotopanaxatrial II was 3:3:1;

[0207] (10) Comparative Example 4 (80mg / kg): The mass ratio of Rh4, F4 and Rk3 was 3:3:1.

[0208] The medication was administered once daily in the morning via gavage, 0.2 ml each time, for 5 consecutive days.

[0209] 2.1.3 Modeling Method

[0210] Except for the control group, all other groups of mice were intraperitoneally injected with 0.1% CCl4 olive oil diluted solution at a volume of 10 ml / kg. The mice were then administered the drug by gavage once 3 hours and once 12 hours after the injection of CCl4 olive oil diluted solution (the drug administration was the same as before modeling).

[0211] 2.1.4 Sample Preparation

[0212] Twenty-four hours after injection of CCl4-ol diluted olive oil, mice were enucleated to collect blood. After standing for 30 minutes, the blood was centrifuged at 3000 r / min to collect serum. Part of the serum was used to measure ALT, AST, MDA, GSH-PX, IL-6, and other indicators (according to the kit instructions), and the rest was used for metabolomics studies. After blood collection, the mice were sacrificed, and the livers were collected, rinsed with physiological saline, and part of the liver was fixed with 4% paraformaldehyde, stained with H&E, and subjected to liver tissue morphology examination. The remaining part of the liver was homogenized and used for metabolomics studies.

[0213] 2.1.5 Data Statistics

[0214] Data are expressed as mean ± standard error (Mean ± SEM). One-way ANOVA was performed using GraphPad Prism 10.1.2, combined with Tukey's multiple comparisons method to assess differences between groups and identify the sources of these differences. The statistical significance level was set at [value missing]. p <0.05.

[0215] 2.2 Experimental Results

[0216] 2.2.1 Effects on serum ALT and AST levels

[0217] Serological test results are shown in Table 9 and Figure 12 As shown, compared with the blank group, the serum ALT and AST levels in the model group mice were significantly increased, sensitively reflecting the degree of liver damage, indicating that the ALI model was successfully established. In the positive drug control group, the serum ALT and AST levels were decreased, showing a significant difference compared with the model group. p <0.01, p <0.01); A combination of three rare ginsenosides reduced the CCl4-induced increase in ALT ( p <0.001, p <0.001, p <0.001) and elevated ALT ( p <0.001, p <0.001, p <0.01), and rare ginsenoside composition 1-rare ginsenoside composition 3 reduced ALT levels ( p <0.01, p <0.05, p <0.05, p <0.05) and AST level ( p <0.05, p <0.05, p <0.05, p The results (<0.05) were significantly better than all comparative groups, indicating that the rare ginsenoside composition can improve the elevated ALT and AST levels caused by the drug CCl4 and play a protective role in the liver.

[0218] Table 9. Serum ALT and AST levels in each group of mice ( ± SEM, n=6)

[0219]

[0220] Note: Compared to the blank group ### p <0.001; compared to the model group, p <0.01, p <0.001; compared to rare ginsenoside composition 3, ▲ p <0.05, ▲▲ p <0.01.

[0221] 2.2.2 Effects on serum MDA levels and SOD activity

[0222] The results of serum MDA levels and SOD activity measurements are shown in Table 10. Figure 13 As can be seen from the data in the table, compared with the normal group, the serum MDA level in the model group mice was significantly increased ( p The value <0.001 indicates that CCl4, while inducing ALI, also altered serum MDA levels. Administration of biphenyl diester drops via gavage significantly reversed the CCl4-induced increase in serum MDA levels. p <0.01). Similarly, group 1 was administered the rare ginsenoside composition by gavage ( p <0.001), two groups of rare ginsenoside compositions ( p <0.001), three groups of rare ginsenoside compositions ( p <0.001), and can also significantly reduce serum MDA levels. Furthermore, rare ginsenoside composition 1-rare ginsenoside composition 3 have an effect on reducing MDA levels ( p <0.001, p <0.01, p <0.01, p <0.001) significantly outperformed all comparative groups.

[0223] As can be seen from the data in the table, compared with the normal group, the activity of SOD in the serum of mice in the model group was significantly reduced. p <0.05, indicating that ALI is closely related to the level of SOD in serum. Oral administration of biphenyl diester drops significantly increased the level of SOD in the serum of liver-injured mice. p <0.05%. It was also observed that group 1, administered via gavage with a high concentration of rare ginsenosides (Group 1), showed improvement. p <0.05), two groups of rare ginsenoside compositions ( p <0.05), three groups of rare ginsenoside compositions ( p <0.05%, it can also significantly increase the SOD level in the serum of ALI mice, and rare ginsenoside composition 1-rare ginsenoside composition 3 can reduce the SOD level ( p <0.05,p <0.05, p <0.05, p <0.05) significantly better than all comparative groups.

[0224] Table 10. Serum levels of MDA and SOD in each group of mice ( ± SEM, n=6)

[0225]

[0226] Note: Compared to the blank group, # p <0.05, ### p <0.001; compared to the model group, p <0.05, p <0.01, p <0.001; compared to rare ginsenoside composition 3, ▲▲ p <0.01, ▲▲▲ p <0.001.

[0227] 2.2.3 Effects on serum ROS and GSH-PX levels

[0228] The results of serum ROS and GSH-PX measurements are shown in Table 11 and... Figure 14 As can be seen from the data in the table, compared with the blank group, the serum ROS level of mice in the model group was significantly increased ( p <0.01). This indicates that CCl4-induced ALI is associated with elevated serum ROS levels. Oral administration of biphenyl diester drops significantly reversed the CCl4-induced increase in serum ROS levels. p <0.05%. Simultaneously, gavage administration of rare ginsenoside composition 1, rare ginsenoside composition 2, and rare ginsenoside composition 3 significantly reduced serum ROS levels in liver-injured mice. p <0.05, p <0.05, p <0.05%. Furthermore, rare ginsenoside composition 1-rare ginsenoside composition 3 reduced ROS levels ( p <0.01, p <0.01, p <0.05, p <0.05) significantly better than all comparative groups.

[0229] As can be seen from the data in the table, compared with the blank group, the serum GSH-PX level in the model group mice was significantly reduced. p <0.001). Oral administration of biphenyl diester drops significantly increased serum GSH-PX levels in liver-injured mice. p <0.001). It was also observed that gavage administration of rare ginsenoside composition group 1, rare ginsenoside composition group 2, and rare ginsenoside composition group 3 significantly increased the serum GSH-PX level in liver-injured mice. p <0.001, p <0.001, p <0.001), and rare ginsenoside composition 1-rare ginsenoside composition 3 reduced ROS levels ( p <0.05, p <0.05, p <0.05, p <0.01) significantly better than all comparative groups.

[0230] Table 11. Serum ROS and GSH-PX levels in each group of mice ( ± SEM, n=6)

[0231]

[0232] Note: Compared to the blank group, ## p <0.01, ### p <0.001; compared to the model group, p <0.05, p <0.001; compared to rare ginsenoside composition 3, ▲ p <0.05, ▲▲ p <0.01.

[0233] 2.2.4 Effect on serum IL-6 levels

[0234] The results of serum IL-6 level measurement are shown in Table 12 and Figure 15 As can be seen from the data in the table, compared with the blank group, the serum IL-6 inflammatory factor level in the model group mice was significantly increased ( p<0.001). This indicates that CCl4 can induce inflammation while causing ALI. Compared with the model group, oral administration of biphenyl diester drops significantly reduced serum IL-6 levels induced by ALI ( p <0.01). It was also observed that gavage administration of rare ginsenoside composition group 1, rare ginsenoside composition group 2, and rare ginsenoside composition group 3 could also reduce the serum IL-6 level in liver-injured mice. p <0.01, p <0.01, p <0.01), and rare ginsenoside composition 1-rare ginsenoside composition 3 reduced ROS levels ( p <0.05, p <0.05, p <0.01, p <0.05) significantly better than all comparative groups.

[0235] Table 12 Serum IL-6 levels in each group of mice ( ± SE.M., n=6)

[0236]

[0237] Note: Compared to the blank group, ### p <0.001; compared to the model group, p <0.01; compared to rare ginsenoside composition 3, ▲ p <0.05, ▲▲ p <0.01.

[0238] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A rare ginsenoside composition, characterized in that, The rare ginsenoside composition consists of Rh4, F4 and Dehydroprotopanaxatrial II, with a mass ratio of (2-4):(2-4):

1.

2. The rare ginsenoside composition according to claim 1, characterized in that, The mass ratio of Rh4, F4 and Dehydroprotopanaxatrial II is 3:3:

1.

3. The method for preparing the rare ginsenoside composition according to any one of claims 1-2, characterized in that, Includes the following steps: (1) Take the total saponin powder of ginseng stems and leaves, add anhydrous ethanol, concentrate under reduced pressure to obtain preliminary ginseng extract; (2) Dissolve the preliminary ginseng extract in step (1), use ether and n-butanol as solvents in sequence, shake to treat, collect the supernatant, concentrate to obtain the secondary refined extract; (3) Add β-glucanase to the extract obtained in step (2), and place the mixture in a shaker, maintain the temperature at 40℃-50℃ and the pH at 6.5-7.5, and carry out the reaction. After the reaction is completed, centrifuge to prepare the preliminary product. (4) The preliminary product was separated and purified by C-18 semi-preparative high performance liquid chromatography to obtain high purity Rh4, F4 and Dehydroprotopanaxatrial II monomers, which were then mixed according to the mass ratio to obtain a rare ginsenoside composition.

4. The use of the rare ginsenoside composition according to any one of claims 1-2 in the preparation of products that relieve physical fatigue.

5. The application according to claim 4, characterized in that, The products mentioned include health supplements and medicines.

6. The application according to claim 5, characterized in that, The product is a drug, and the drug has at least one of the following effects: (1) Relieve physical fatigue; (2) Reduce serum urea nitrogen content and decrease protein breakdown for energy; (3) Increase liver glycogen levels and improve fatigue resistance.

7. The application according to claim 5, characterized in that, When the product is a drug, the drug includes a pharmaceutically acceptable carrier.

8. The use of the rare ginsenoside composition according to any one of claims 1-2 in the preparation of a medicament for relieving or treating alcoholic liver injury.

9. The application according to claim 8, characterized in that, The drug has at least one of the following effects: (1) Reduce serum ALT and AST levels; (2) Increase the levels of GSH and SOD in liver tissue; (3) Reduces MDA levels in liver tissue; (4) Reduces TG levels in liver tissue; (5) Improves the degree of inflammatory infiltration of hepatocytes; (6) Reduce serum ROS levels; (7) Increase serum GSH-PX levels; (8) Reduce serum IL-6 levels.

Images