Pharmaceutical composition, preparation method thereof and application in preparing anti-fatigue drugs

Abstract

The present invention provides a pharmaceutical composition, a preparation method thereof, and an application thereof in the preparation of an anti-fatigue drug, relating to the technical field of pharmaceutical compositions. The pharmaceutical composition comprises rare ginsenoside RK3 and taurine. The rare ginsenoside RK3 and taurine are used in combination as a pharmaceutical composition. The components are simple and easy to obtain. The combined use of rare ginsenoside RK3 and taurine has a strong synergistic effect, can significantly relieve physical fatigue, and has a significant anti-fatigue effect. The rare ginsenoside RK3 and taurine drugs have a synergistic effect when the weight ratio is (0.5 to 2): (0.5 to 3), can synergistically increase the time of mice swimming with weight, can synergistically reduce the urea nitrogen content in the serum of mice after swimming with weight, can synergistically reduce the lactic acid content in the serum of mice after swimming with weight, and can synergistically reduce the consumption of glycogen in mice during swimming with weight.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical compositions, and in particular to a pharmaceutical composition, a preparation method thereof, and application thereof in the preparation of anti-fatigue drugs. Background Art

[0002] Fatigue is a temporary state of decreased physiological and psychological function that occurs after prolonged physical or mental activity. Fatigue manifests as increased energy expenditure, decreased physical and cognitive abilities, slower reaction times, and difficulty concentrating. Fatigue is often accompanied by drowsiness, weakness, lack of energy, and a resistance to continued activity. Fighting fatigue, improving physiological adaptability, and improving athletic ability have become common pursuits for improving the quality of life and practical life in modern society. Fatigue is a subjective feeling of discomfort in the human body, but it objectively deprives individuals of the ability to complete normal activities and work under the same conditions. Fighting fatigue involves eliminating fatigue through specific methods / means, allowing individuals to feel relaxed and resume normal activities and / or work.

[0003] Currently, there are many anti-fatigue pharmaceutical compositions, most of which are Chinese herbal medicines with anti-fatigue properties. However, the existing pharmaceutical compositions have many components and have poor anti-fatigue effects. Summary of the Invention

[0004] The problem solved by the present invention is how to solve the problem that the existing pharmaceutical composition has many components and poor anti-fatigue effect.

[0005] To solve the above problems, the present invention provides a pharmaceutical composition, a preparation method thereof, and an application of the pharmaceutical composition in preparing anti-fatigue drugs.

[0006] In a first aspect, the present invention provides a pharmaceutical composition comprising rare ginsenoside RK3 and taurine, wherein the weight ratio of rare ginsenoside RK3 to taurine is (0.5 to 2): (0.5 to 3).

[0007] Optionally, the weight ratio of rare ginsenoside RK3 to taurine is (1 to 1.5):(1 to 2.5).

[0008] Optionally, the weight ratio of rare ginsenoside RK3 to taurine is 1.5:(2 to 3).

[0009] Optionally, the weight ratio of rare ginsenoside RK3 to taurine is 1.5:2.

[0010] In a second aspect, the present invention provides a method for preparing a pharmaceutical composition, which is used to prepare the pharmaceutical composition as described in any one of the above items, comprising the steps of: combining rare ginsenoside RK3 and taurine.

[0011] Optionally, rare ginsenoside RK3 is obtained by the following steps:

[0012] Ginseng slices are taken after cutting, and inoculated onto a culture medium for cultivation, so that callus tissue forms on the surface of the ginseng slices;

[0013] The inducer is added and the culture is continued. Under the action of the inducer, the ginsenoside precursor is converted into ginsenoside RK3;

[0014] Add methanol or ethanol and extract by ultrasonication;

[0015] Solid impurities were removed to obtain a crude extract, which was separated and purified by column chromatography to obtain rare ginsenoside RK3.

[0016] Optionally, the culture medium comprises MS medium, a plant growth regulator, sucrose and agar powder; the plant growth regulator is 2,4-dichlorophenoxyacetic acid and / or 6-benzylaminopurine.

[0017] Optionally, the inducer is methyl jasmonate, and the induction concentration of methyl jasmonate is 100 to 200 μmol / L.

[0018] Optionally, the column chromatography method uses a silica gel column and performs gradient elution with a chloroform-methanol mixed solvent.

[0019] In a third aspect, the present invention provides a use of the pharmaceutical composition as described above in the preparation of an anti-fatigue drug.

[0020] The pharmaceutical composition of the present invention, its preparation method, and its use in preparing anti-fatigue drugs have the following beneficial effects: rare ginsenoside RK3 and taurine are used in combination as the pharmaceutical composition, and the rare ginsenoside RK3 and taurine drugs have a synergistic effect when the weight ratio is (0.5 to 2): (0.5 to 3), showing significant synergistic synergy in anti-fatigue, so that the pharmaceutical composition has a significant anti-fatigue effect, can synergistically increase the time of weighted swimming in mice, can synergistically reduce the urea nitrogen content in the serum of mice after weighted swimming, can synergistically reduce the lactic acid content in the serum of mice after weighted swimming, and can synergistically reduce the consumption of liver glycogen in mice during weighted swimming. DETAILED DESCRIPTION

[0021] To make the above-mentioned objects, features, and advantages of the present invention more clearly understood, specific embodiments of the present invention are described in detail below. It should be understood that the present invention can be implemented in various forms and should not be construed as limited to the embodiments described herein. Instead, these embodiments are provided to provide a more thorough and complete understanding of the present invention. It should be understood that the embodiments of the present invention are intended for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0022] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the present invention description are only for the purpose of describing specific embodiments and are not intended to limit the present invention;

[0023] As used herein, the term "including" and its variations are open-ended, i.e., "including but not limited to"; the term "based on" means "based at least in part on"; the term "one embodiment" means "at least one embodiment"; the term "another embodiment" means "at least one additional embodiment"; the term "some embodiments" means "at least some embodiments"; and the term "optionally" means "optional embodiments." Definitions of other terms are provided in the following description.

[0024] In the related art, ginsenoside RK3 is a ginsenoside extracted from ginseng. The main difference between ginsenoside RK3 and rare ginsenoside RK3 lies in their sources and biological activities. Ginsenoside RK3 is a ginsenoside extracted directly from plants of the Araliaceae family (such as American ginseng, ginseng, Panax notoginseng, etc.) and belongs to the prototype ginsenoside. Rare ginsenoside RK3 is the active ingredient formed after the prototype ginsenoside RK3 is treated with a specific enzyme or subjected to a long period of metabolic conversion, and has stronger biological activity. Rare ginsenoside RK3 is a rare ingredient in the ginsenoside family, also known as ginseng rare saponin RK3, which has anti-fatigue effects. It can delay the occurrence of fatigue by promoting energy metabolism and improving the body's endurance and resistance. Taurine is a multifunctional amino acid derivative with important physiological effects, especially in anti-oxidation, energy metabolism, neuroprotection and cardiovascular health. It is particularly prominent in anti-fatigue and physical fitness enhancement, and is therefore commonly found in health products and functional beverages.

[0025] In response to the problems existing in the above-mentioned related technologies, embodiments of the present invention provide a pharmaceutical composition, a preparation method thereof, and an application of the pharmaceutical composition in the preparation of anti-fatigue drugs.

[0026] An embodiment of the present invention provides a pharmaceutical composition comprising rare ginsenoside RK3 and taurine, wherein the weight ratio of rare ginsenoside RK3 to taurine is (0.5 to 2):(0.5 to 3).

[0027] In this embodiment, rare ginsenoside RK3 and taurine are used in combination as a pharmaceutical composition. The rare ginsenoside RK3 and taurine drugs have a synergistic effect when the weight ratio is (0.5 to 2): (0.5 to 3), showing significant synergistic effect in anti-fatigue (p < 0.05), so that the pharmaceutical composition has a significant anti-fatigue effect, can synergistically increase the time of mice swimming with weight, can synergistically reduce the urea nitrogen content in the serum of mice after swimming with weight, can synergistically reduce the lactic acid content in the serum of mice after swimming with weight, can synergistically reduce the consumption of glycogen in mice during swimming with weight. If taurine and rare ginsenoside RK3 in the pharmaceutical composition are not within the specified ratio range, then there is no synergistic effect (p> 0.05).

[0028] Specifically, the rare ginsenoside RK3 refers to the compound described in the following Chemical Formula 1.

[0029] Chemical formula 1

[0030]

[0031] Taurine refers to the compound represented by the following Chemical Formula 2.

[0032] Chemical formula 2

[0033]

[0034] Specifically, taurine can be produced by the reaction of ethanolamine and sodium bisulfite.

[0035] Optionally, the weight ratio of rare ginsenoside RK3 to taurine is (1 to 1.5):(1 to 2.5).

[0036] In this optional embodiment, the pharmaceutical composition of taurine and rare ginsenoside RK3 are combined within a limited ratio range, and the synergistic effect is more obvious, with a significant anti-fatigue effect.

[0037] Optionally, the weight ratio of rare ginsenoside RK3 to taurine is 1.5:(2 to 3).

[0038] Optionally, the weight ratio of rare ginsenoside RK3 to taurine is 1.5:2.

[0039] An embodiment of the present invention provides a method for preparing a pharmaceutical composition, which is used to prepare the pharmaceutical composition as described in any one of the above items, comprising the steps of: combining rare ginsenoside RK3 and taurine.

[0040] Optionally, rare ginsenoside RK3 is obtained by the following steps:

[0041] Ginseng slices are taken after cutting, and inoculated onto a culture medium for cultivation, so that callus tissue forms on the surface of the ginseng slices;

[0042] The inducer is added and the culture is continued. Under the action of the inducer, the ginsenoside precursor is converted into ginsenoside RK3;

[0043] Add methanol or ethanol and extract by ultrasonication;

[0044] Solid impurities were removed to obtain a crude extract, which was separated and purified by column chromatography to obtain rare ginsenoside RK3.

[0045] Specifically, the culture medium includes MS culture medium, plant growth regulator, sucrose and agar powder; the plant growth regulator is 2,4-dichlorophenoxyacetic acid and / or 6-benzylaminopurine.

[0046] Specifically, the inducer is methyl jasmonate, and the induction concentration of methyl jasmonate is 100 to 200 μmol / L.

[0047] Optionally, the column chromatography method uses a silica gel column and performs gradient elution with a chloroform-methanol mixed solvent.

[0048] It can be understood that the details of the preparation method of the pharmaceutical composition in this embodiment may include:

[0049] 1. Selection and disinfection of explants

[0050] (1) Explant selection: Ginseng roots, stems, and leaves are usually selected as explants. Ginseng roots contain a large amount of ginsenoside precursors and are ideal materials. For example, select healthy, pest-free ginseng roots and cut them into small segments, each approximately 0.5 to 1 cm in length.

[0051] (2) Disinfection: Rinse the selected explants with running water for 30 to 60 minutes to remove surface dirt and some microorganisms. Then, soak them in 70% to 75% alcohol for 30 to 60 seconds for surface disinfection. Then, soak them in 0.1% to 0.2% mercuric chloride solution for 8 to 10 minutes. Finally, rinse them with sterile water 3 to 5 times to ensure that the microorganisms on the surface of the explants are completely removed.

[0052] 2. Callus induction

[0053] (1) Culture medium preparation: The culture medium commonly used for callus induction is MS medium (Murashige and Skoog medium). Appropriate plant growth regulators are added to the culture medium, such as 2,4-D (2,4-dichlorophenoxyacetic acid), with a concentration of about 1 to 2 mg / L, which can induce explant dedifferentiation to form callus; 6-BA (6-benzylaminopurine) is also added at a concentration of about 0.5 to 1 mg / L to regulate cell division and differentiation. At the same time, the culture medium must contain sucrose as a carbon source at a concentration of about 30 g / L, and agar powder to solidify the culture medium at a concentration of 7 to 8 g / L.

[0054] (2) Culture Conditions: Sterilized explants are inoculated onto induction medium and cultured in a room maintained at 22 to 25°C, with a light intensity of 1000 to 2000 lx and a photoperiod of 12 to 16 hours per day. After approximately 2 to 3 weeks, callus tissue begins to form on the surface of the explants. Callus tissue is generally light yellow or white in color and has a loose texture.

[0055] 3. Cell suspension culture (optional step)

[0056] (1) Establishment of suspension system: If a large number of cells are required to produce ginsenoside RK3, the induced callus can be transferred to a liquid medium for suspension culture. The composition of the liquid medium is similar to that of the solid medium, but does not contain agar powder. The callus is cut into small pieces and placed in a triangular flask containing 50 to 100 ml of liquid medium, with an inoculum size of approximately 3 to 5 g per flask. The culture is carried out on a shaker with a speed of generally 100 to 120 r / min. The temperature and light conditions are the same as those for the callus induction stage.

[0057] (2) Cell proliferation: After a period of culture, cells will continue to proliferate, making the culture medium turbid. During this process, samples should be taken regularly to observe the growth status of cells, generally every 3 to 5 days, and the cell density should be detected by tools such as a blood cell counting plate. When the cell density reaches a certain level, for example, the number of cells per milliliter of culture medium reaches 1×10 6 to 1×10 7 When the cells are fully grown, the next step of cultivation can be carried out.

[0058] 4. Induced synthesis of rare ginsenoside RK3

[0059] (1) Adding inducers: Add substances that can induce the synthesis of rare ginsenoside RK3 to callus tissue or suspension culture cells. For example, methyl jasmonate (MeJA) is a commonly used inducer, with a concentration generally ranging from 100 to 200 μmol / L. It can activate secondary metabolic pathways in cells and promote the synthesis of rare ginsenoside RK3.

[0060] (2) Extending the culture period: After adding the inducer, continue culturing the cells or callus. The culture period may be extended depending on the actual situation and may take about 4 to 6 weeks. During this process, under the action of the inducer, the cells will gradually convert the ginsenoside precursor into the rare ginsenoside RK3.

[0061] 5. Extraction and separation

[0062] (1) Extraction method: After the culture is completed, the cells or callus tissue is collected and extracted using a suitable solvent. Commonly used extraction solvents are methanol or ethanol. The material and the solvent are mixed in a ratio of 1:10 to 1:20 (w / v). Ultrasonic extraction is performed in an ultrasonic cleaner for 30 to 60 minutes at an ultrasonic power of 200 to 300W. Ultrasonic extraction can destroy the cell structure and better dissolve the rare ginsenoside RK3 in the solvent.

[0063] (2) Separation and purification: The extracted solution is filtered or centrifuged (rotation speed of 3000 to 5000 r / min, time of 10 to 15 minutes) to remove solid impurities to obtain a crude extract containing a variety of ginsenosides. Column chromatography, such as silica gel column chromatography, can then be used for separation and purification. The crude extract is loaded onto a silica gel column and gradient eluted with a chloroform-methanol mixed solvent of different proportions. Based on the polarity and other properties of the rare ginsenoside RK3, the eluate containing the target compound is collected and further purified and identified by methods such as high performance liquid chromatography (HPLC), ultimately obtaining a high-purity rare ginsenoside RK3.

[0064] An embodiment of the present invention provides a use of the above-mentioned pharmaceutical composition in the preparation of an anti-fatigue drug.

[0065] The pharmaceutical composition of the present invention can be administered to a subject to combat fatigue. The subject can be a mammal, such as a human, rat, rabbit, sheep, pig, cow, cat, dog, monkey, or the like.

[0066] The present invention is further described below with reference to specific embodiments.

[0067] Example 1. Effects of rare ginsenoside RK3, taurine, and the combination of rare ginsenoside RK3 and taurine on the exhaustive swimming time of mice.

[0068] The compositions were prepared by uniformly mixing rare ginsenoside RK3 and taurine in weight ratios of 0.2:0.5, 0.5:0.5, 0.5:3, 1:1.5, 1:3, 1.5:1, 1.5:2, 1.5:3, 2:1, 2:3, 2:4, 3:1, and 3:3, and were referred to as Composition 1, Composition 2, Composition 3, Composition 4, Composition 5, Composition 6, Composition 7, Composition 8, Composition 9, Composition 10, Composition 11, Composition 12, and Composition 13, respectively.

[0069] Anti-fatigue experiments were conducted on mice using rare ginsenoside RK3, taurine, and Compositions 1 through 13. The daily dosage for a 60-kg adult (1.2 g / 60 kg body weight) is 1.2 g / 60 kg body weight. BW (body weight) represents body weight. For example, mg / kg BW refers to the amount of drug administered per kilogram of body weight.

[0070] Animal source: 160 male Kunming mice, weighing 18-22 g, provided by the Animal Center of Xi'an Jiaotong University. All Kunming mice were observed for 3 days before the experiment and had normal activities.

[0071] Dosage selection: The experimental group was set at 200 mg / kg BW, which was 10 times the daily adult intake. There were 16 groups in total, namely the rare ginsenoside RK3 group, the taurine group, the combination groups 1 to 13, and the blank control group. Each group had 10 mice. The control group was given an equal volume of blank solution. The gavage volume for each mouse was 0.2 ml / 10 g, and the mice were gavaged once a day for 30 consecutive days.

[0072] Exhaustive swimming test: Different drug-treated groups were gavaged once daily for 30 consecutive days. Mice were fasted for 4 hours before the experiment (fatigue-related indicators during acute exercise under fasting conditions can reflect true physiological regulation). A lead wire weight (5% of body weight) was placed on the tail of the mice and placed in a 40 cm deep pool (65 cm high x 20 cm diameter) at 34°C ± 1°C. The mice were forced to swim. The swimming time was recorded. The mice were instructed to swim until their nostrils could not surface within 10 seconds. The exhaustive swimming time was considered the time the mice reached. The exhaustive swimming time for each experimental group is shown in Table 1.

[0073] Table 1 Effects of various compositions on the exhaustive swimming time of mice

[0074]

[0075]

[0076] Note: Data are shown as mean ± standard deviation, where - indicates the group without rare ginsenoside RK3 and taurine, and the control group was used as the basis and not compared with itself, so the p field is blank; * indicates p < 0.05 compared with the control group, ** indicates p < 0.01 compared with the control group, and *** indicates p < 0.001 compared with the control group; # indicates p < 0.05 compared with the rare ginsenoside RK3 group, ## indicates p < 0.01 compared with the rare ginsenoside RK3 group, and ### indicates p < 0.001 compared with the rare ginsenoside RK3 group; & indicates p < 0.05 compared with the taurine group, && indicates p < 0.01 compared with the taurine group, and &&& indicates p < 0.001 compared with the taurine group.

[0077] As can be seen from Table 1, after 30 days of oral administration, the mice in each test group were subjected to an exhaustive swimming test. Compared with the control group, the swimming time of the drug-treated groups was significantly increased (p<0.05), compositions 4 to 8 showed an extremely significant increase compared with the control group (p<0.001), compositions 2 to 9 showed significant differences compared with the rare ginsenoside RK3 and taurine alone (p<0.05), and composition 7 showed an extremely significant difference compared with the rare ginsenoside RK3 and taurine alone (p<0.001), indicating that rare ginsenoside RK3 and taurine drugs can synergistically increase the weighted swimming time of mice within a certain ratio range. There was no significant difference in compositions 11 to 13, indicating that they are outside the scope of the present invention and have no synergistic enhancing effect.

[0078] Example 2: Effects of rare ginsenoside RK3, taurine, and the combination of rare ginsenoside RK3 and taurine on various serum indicators of mice after exercise.

[0079] As in Example 1, the compositions were prepared by uniformly mixing rare ginsenoside RK3:taurine in weight ratios of 0.2:0.5, 0.5:0.5, 0.5:3, 1:1.5, 1:3, 1.5:1, 1.5:2, 1.5:3, 2:1, 2:3, 2:4, 3:1, and 3:3, and were referred to as Composition 1, Composition 2, Composition 3, Composition 4, Composition 5, Composition 6, Composition 7, Composition 8, Composition 9, Composition 10, Composition 11, Composition 12, and Composition 13, respectively.

[0080] Experiments were conducted using rare ginsenoside RK3, taurine, and Compositions 1 through 13 to investigate the effects of various serum markers on mice after exercise. The daily dosage for an adult (60 kg) of these compositions is 1.2 g / 60 kg body weight (BW). BW stands for body weight. For example, mg / kg BW refers to milligrams of drug per kilogram of body weight.

[0081] Animal source: 160 male Kunming mice, weighing 18-22 g, provided by the Animal Center of Xi'an Jiaotong University. All Kunming mice were observed for 3 days before the experiment and had normal activities.

[0082] Dosage selection: The experimental group was set at 200 mg / kg BW, which was 10 times the daily adult intake. There were 16 groups in total, namely the rare ginsenoside RK3 group, the taurine group, the combination groups 1 to 13, and the blank control group. Each group had 10 mice. The control group was given an equal volume of blank solution. The gavage volume for each mouse was 0.2 ml / 10 g, and the mice were gavaged once a day for 30 consecutive days.

[0083] (1) Determination of serum urea nitrogen content:

[0084] Different drug treatment groups were gavaged once a day for 30 consecutive days. Before the experiment, mice were fasted for 4 hours (under fasting conditions, fatigue-related indicators during acute exercise can reflect the true physiological regulation ability). The mice were then loaded with a lead wire weighing 5% of their body weight on their tails and placed in a 40cm deep swimming pool (65cm high * 20cm diameter) at a water temperature of 34℃±1℃ for forced swimming. During the forced swimming period, the mice rested for 60 minutes, their eyeballs were removed, and about 1.0mL of whole blood (without anticoagulant) was collected and placed in a 4℃ refrigerator for about 60 minutes. After the blood coagulated, it was centrifuged at 3000rpm / min for 15 minutes. The serum was collected for later use and the urea nitrogen content in the serum was determined using a kit from Boyan Biotechnology (see Table 2).

[0085] Table 2 Effects of various compositions on serum urea nitrogen content in mice after exhaustive swimming

[0086]

[0087]

[0088] Note: Data are shown as mean ± standard deviation, where - indicates the group without rare ginsenoside RK3 and taurine, and the control group was used as the basis and not compared with itself, so the p field is blank; * indicates p < 0.05 compared with the control group, ** indicates p < 0.01 compared with the control group, and *** indicates p < 0.001 compared with the control group; # indicates p < 0.05 compared with the rare ginsenoside RK3 group, ## indicates p < 0.01 compared with the rare ginsenoside RK3 group, and ### indicates p < 0.001 compared with the rare ginsenoside RK3 group; & indicates p < 0.05 compared with the taurine group, && indicates p < 0.01 compared with the taurine group, and &&& indicates p < 0.001 compared with the taurine group.

[0089] As can be seen from Table 2, after 30 days of oral administration, the mice in each test group were subjected to an exhaustive swimming test. Compared with the control group, the rare ginsenoside RK3, taurine and combination groups all reduced the urea content in the mouse serum. The effect of composition 2 to 10 groups on reducing serum urea nitrogen content was more significant (p < 0.05). Compared with the rare ginsenoside RK3 group, the effect of composition 2 to 10 on reducing the serum urea nitrogen of mice was more significant than that of the rare ginsenoside RK3 group (p < 0.05), among which composition 7 had a stronger ability to reduce the serum urea nitrogen of mice than the rare ginsenoside RK3 group (p < 0.01). Compared with the taurine group, the effect of composition 2 to 10 on reducing the serum urea nitrogen of mice was more significant than that of the taurine group (p < 0.05), among which composition 7 had a stronger ability to reduce the serum urea nitrogen of mice than the taurine group (p < 0.01). The results indicate that rare ginsenoside RK3 and taurine drugs can synergistically reduce the urea nitrogen content in the serum of mice after weighted swimming within a certain ratio range, among which composition 7 has a more significant effect, and compositions 11 to 13 have no obvious differences, indicating that they are outside the scope of the present invention and have no obvious synergistic effect, but they still have the effect of reducing the urea nitrogen content in the serum of mice.

[0090] (2) Determination of blood lactate content

[0091] Different drug-treated groups were gavaged once daily for 28 consecutive days. Mice were fasted for 4 hours before the experiment (fatigue-related indicators during acute exercise under fasting conditions can reflect true physiological regulation). A lead wire 5% of their body weight was placed on their tails and placed in a 40cm deep pool (65cm high x 20cm diameter) at 34°C ± 1°C for forced swimming. During the forced swimming period, the mice rested for 60 minutes. Their eyes were then removed and approximately 1.0mL of whole blood (without anticoagulant) was collected from the mice and placed in a 4°C refrigerator for approximately 60 minutes. After the blood coagulated, it was centrifuged at 3000 rpm / min for 15 minutes. Serum was then collected and used for measurement of blood lactate using a kit from Boyan Biotechnology.

[0092] The statistical results are shown in Table 3 below:

[0093] Table 3 Effects of various compositions on blood lactate content in mice after exhaustive swimming

[0094]

[0095]

[0096] Note: Data are shown as mean ± standard deviation, where - indicates the group without rare ginsenoside RK3 and taurine, and the control group was used as the basis and not compared with itself, so the p field is blank; * indicates p < 0.05 compared with the control group, ** indicates p < 0.01 compared with the control group, and *** indicates p < 0.001 compared with the control group; # indicates p < 0.05 compared with the rare ginsenoside RK3 group, ## indicates p < 0.01 compared with the rare ginsenoside RK3 group, and ### indicates p < 0.001 compared with the rare ginsenoside RK3 group; & indicates p < 0.05 compared with the taurine group, && indicates p < 0.01 compared with the taurine group, and &&& indicates p < 0.001 compared with the taurine group.

[0097] As can be seen from Table 3, after 28 days of oral administration, the mice in each test group were subjected to an exhaustive swimming test. Compared with the control group, the rare ginsenoside RK3, taurine and combination groups all reduced the lactic acid content in the mouse serum. Compared with the rare ginsenoside RK3 group, compositions 2 to 10 had a more significant effect on reducing the serum lactic acid of mice than the rare ginsenoside RK3 group (p < 0.05), among which composition 7 had a stronger ability to reduce the serum lactic acid of mice than the rare ginsenoside RK3 group (p < 0.01). Compared with the taurine group, compositions 2 to 10 had a more significant effect on reducing the serum lactic acid of mice than the taurine group (p < 0.05), among which composition 7 had a stronger ability to reduce the serum lactic acid of mice than the taurine group (p < 0.01). The results indicate that rare ginsenoside RK3 and taurine drugs can synergistically reduce the lactic acid content in the serum of mice after weighted swimming within a certain ratio range, among which composition 7 has a more significant effect, and compositions 11 to 13 have no obvious differences, indicating that the compositions outside the scope of the present invention have no obvious synergistic effect, but they still have the effect of reducing lactic acid in mouse serum.

[0098] Example 3. Effects of rare ginsenoside RK3, taurine, and the combination of rare ginsenoside RK3 and taurine on liver glycogen in mice after exercise.

[0099] As in Example 1, the compositions were prepared by uniformly mixing rare ginsenoside RK3:taurine in weight ratios of 0.2:0.5, 0.5:0.5, 0.5:3, 1:1.5, 1:3, 1.5:1, 1.5:2, 1.5:3, 2:1, 2:3, 2:4, 3:1, and 3:3, and were referred to as Composition 1, Composition 2, Composition 3, Composition 4, Composition 5, Composition 6, Composition 7, Composition 8, Composition 9, Composition 10, Composition 11, Composition 12, and Composition 13, respectively.

[0100] Experiments were conducted using rare ginsenoside RK3, taurine, and Compositions 1 through 13 to investigate the effects of various serum markers on mice after exercise. The daily dosage for an adult (60 kg) of these compositions is 1.2 g / 60 kg body weight (BW). BW stands for body weight. For example, mg / kg BW refers to milligrams of drug per kilogram of body weight.

[0101] Animal source: 160 male Kunming mice, weighing 18-22 g, provided by the Animal Center of Xi'an Jiaotong University. All Kunming mice were observed for 3 days before the experiment and had normal activities.

[0102] Dosage selection: The experimental group was set at 200 mg / kg BW, which was 10 times the daily adult intake. There were 16 groups in total, namely the rare ginsenoside RK3 group, the taurine group, the combination groups 1 to 13, and the blank control group. Each group had 10 mice. The control group was given an equal volume of blank solution. The gavage volume for each mouse was 0.2 ml / 10 g, and the mice were gavaged once a day for 30 consecutive days.

[0103] The different drug-treated groups were gavaged once daily for 28 consecutive days. Mice were fasted for 4 hours before the experiment (fatigue-related indicators during acute exercise under fasting conditions can reflect true physiological regulation). A lead wire (5% of body weight) was placed on the tails of the mice and they were placed in a 40 cm deep pool (65 cm high x 20 cm diameter) at 34°C ± 1°C for forced swimming. Forced swimming, the mice rested for 60 minutes, and after being killed by cervical dislocation, the liver was removed, rinsed with physiological saline, and dried with filter paper. 200 mg of liver was accurately weighed, and 8 mL of trichloroacetic acid was added. Each tube was homogenized for 1 minute, and the homogenate was poured into a centrifuge tube and centrifuged at 3000 rpm for 15 minutes. The supernatant was transferred to another test tube, 1 mL of supernatant was placed in a 10-well centrifuge tube, 4 mL of 95% ethanol was added, and the mixture was thoroughly mixed and placed in a 37 to 40°C water bath for 3 hours. The test tube was centrifuged at 3000 rpm for 15 minutes. The supernatant was carefully discarded and the test tube was inverted for 10 minutes before the precipitate was taken. The anthrone method was used to determine the liver glycogen content.

[0104] The statistical results are shown in Table 4 below:

[0105] Table 4 Effects of various compositions on liver glycogen content in mice after exhaustive swimming

[0106]

[0107]

[0108] Note: Data are shown as mean ± standard deviation, where - indicates the group without rare ginsenoside RK3 and taurine, and the control group was used as the basis and not compared with itself, so the p field is blank; * indicates p < 0.05 compared with the control group, ** indicates p < 0.01 compared with the control group, and *** indicates p < 0.001 compared with the control group; # indicates p < 0.05 compared with the rare ginsenoside RK3 group, ## indicates p < 0.01 compared with the rare ginsenoside RK3 group, and ### indicates p < 0.001 compared with the rare ginsenoside RK3 group; & indicates p < 0.05 compared with the taurine group, && indicates p < 0.01 compared with the taurine group, and &&& indicates p < 0.001 compared with the taurine group.

[0109] As shown in Table 4, after 28 days of oral administration, the mice in each test group were subjected to an exhaustive swimming test. Compared with the control group, the liver glycogen consumption of the mice in the rare ginsenoside RK3, taurine and combination groups was significantly reduced (p < 0.05). Compared with the rare ginsenoside RK3 group, the liver glycogen consumption of compositions 2 to 10 was significantly lower than that of the rare ginsenoside RK3 group (p < 0.05), among which the liver glycogen consumption of mice in compositions 4 to 9 was extremely significantly reduced (p < 0.01). Compared with the taurine group, the liver glycogen consumption of mice in compositions 2 to 10 was significantly reduced compared with the taurine group (p < 0.05), among which composition 7 extremely significantly reduced the liver glycogen consumption of mice (p < 0.001). This indicates that rare ginsenoside RK3 and taurine drugs can synergistically reduce the consumption of liver glycogen in mice during weighted swimming within a certain ratio range, among which composition 7 has a more significant effect, and compositions 11 to 13 have no obvious differences from the rare ginsenoside group and the taurine group, indicating that outside the scope of the present invention, the composition has no obvious synergistic effect, but it still has the effect of reducing serum lactic acid in mice.

[0110] Although the present invention is disclosed as above, the protection scope of the present invention is not limited thereto. Those skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention, and these changes and modifications will fall within the protection scope of the present invention.

Claims

1. A pharmaceutical composition for anti-fatigue, characterized in that: The invention is composed of rare ginsenoside RK3 and taurine, wherein the weight ratio of the rare ginsenoside RK3 to the taurine is 1.5:(2 to 3).

2. The anti-fatigue pharmaceutical composition according to claim 1, characterized in that The weight ratio of the rare ginsenoside RK3 to the taurine is 1.5:

2.

3. A method for preparing a pharmaceutical composition for anti-fatigue, characterized in that: The method for preparing the anti-fatigue pharmaceutical composition according to any one of claims 1 to 2 comprises the steps of combining rare ginsenoside RK3 and taurine.

4. The method for preparing the anti-fatigue pharmaceutical composition according to claim 3, characterized in that: The rare ginsenoside RK3 is obtained by the following steps: Taking ginseng slices after cutting, inoculating them onto a culture medium for cultivation, and forming callus tissue on the surface of the ginseng slices; adding an inducer and continuing the culture; under the action of the inducer, the ginsenoside precursor is converted into ginsenoside RK3; Add methanol or ethanol and extract by ultrasonication; Solid impurities are removed to obtain a crude extract, which is separated and purified by column chromatography to obtain the rare ginsenoside RK3.

5. The method for preparing the anti-fatigue pharmaceutical composition according to claim 4, characterized in that: The culture medium comprises MS culture medium, plant growth regulator, sucrose and agar powder; the plant growth regulator is 2,4-dichlorophenoxyacetic acid and / or 6-benzylaminopurine.

6. The method for preparing the anti-fatigue pharmaceutical composition according to claim 4, characterized in that: The inducer is methyl jasmonate, and the induction concentration of the methyl jasmonate is 100 to 200 μmol / L.

7. The method for preparing the anti-fatigue pharmaceutical composition according to claim 4, characterized in that: The column chromatography method uses a silica gel column and performs gradient elution with a chloroform-methanol mixed solvent.

8. Use of the anti-fatigue pharmaceutical composition according to any one of claims 1 to 2 in the preparation of anti-fatigue drugs.

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