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

Abstract

The invention provides a pharmaceutical composition as well as a preparation method and application thereof in preparation of an anti-fatigue medicine, and relates to the technical field of pharmaceutical compositions. The pharmaceutical composition comprises rare ginsenoside RK3 and taurine. The rare ginsenoside RK3 and the taurine are combined to serve as the pharmaceutical composition, the components are simple and easy to obtain, the rare ginsenoside RK3 and the taurine are combined to have a strong synergistic effect, physical fatigue can be remarkably relieved, and the pharmaceutical composition has a remarkable anti-fatigue effect. The rare ginsenoside RK3 and the taurine have a synergistic effect when the weight ratio of the rare ginsenoside RK3 to the taurine is (0.5-2): (0.5-3), can synergistically prolong the time of mouse weight-attached swimming, can synergistically reduce the content of urea nitrogen in serum of the mouse after weight-attached swimming, can synergistically reduce the content of lactic acid in serum of the mouse after weight-attached swimming, and can improve the immunity of the mouse. The consumption of hepatic glycogen in the mouse weight-attached swimming can be synergistically reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical compositions, and in particular to a pharmaceutical composition and 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 after prolonged physical or mental activity. Fatigue manifests itself as increased energy consumption, weakened physical and cognitive abilities, slower reaction speeds, and difficulty concentrating. Fatigue is usually accompanied by drowsiness, weakness, lack of energy, and resistance to continued activity. Fighting fatigue, improving physiological adaptability, and improving athletic ability have become common pursuits of the people in modern society to improve their quality of life and life practices. Fatigue is a subjective discomfort of the human body, but it objectively causes individuals to lose their ability to complete normal activities and work under the same conditions. Fighting fatigue is to eliminate fatigue through certain methods / means, so that individuals feel relaxed and can resume normal activities and / or work.

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

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

[0005] In order to solve the above problems, the present invention provides a pharmaceutical composition and a preparation method thereof and application thereof in the preparation of 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 and 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] Taking ginseng slices after cutting, inoculating them on a culture medium for cultivation, and forming callus tissue 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 beneficial effects of the pharmaceutical composition of the present invention, the preparation method thereof and the use thereof in the preparation of anti-fatigue drugs are as follows: rare ginsenoside RK3 and taurine are used in combination as the 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), and significant synergistic effect is shown in anti-fatigue, so that the pharmaceutical composition has significant anti-fatigue effect, can synergistically increase the weighted swimming time of 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 glycogen in mice during weighted swimming. DETAILED DESCRIPTION

[0021] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the 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 being 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 only used for exemplary purposes 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 specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention;

[0023] The term "including" and its variations used herein are open inclusions, 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 other embodiment"; the term "some embodiments" means "at least some embodiments"; the term "optionally" means "optional embodiments". The relevant definitions of other terms will be given 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 is their source and biological activity. Ginsenoside RK3 is a ginsenoside extracted directly from Araliaceae plants (such as American ginseng, ginseng, Panax notoginseng, etc.), and belongs to the prototype ginsenoside. Rare ginsenoside RK3 is an active ingredient formed after prototype ginsenoside RK3 is treated with a specific enzyme or subjected to a long period of metabolic transformation, 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 an anti-fatigue effect. 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, so it is commonly found in health products and functional beverages.

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

[0026] A pharmaceutical composition provided by an embodiment of the present invention comprises 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. Rare ginsenoside RK3 and taurine drugs have a synergistic effect when the weight ratio is (0.5 to 2): (0.5 to 3), and show 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, and 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 by the following Chemical Formula 1.

[0029] Chemical formula 1

[0030]

[0031] Taurine refers to a 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 and 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 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] A method for preparing a pharmaceutical composition provided in an embodiment of the present invention 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] Taking ginseng slices after cutting, inoculating them on a culture medium for cultivation, and forming callus tissue 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 ultrasonic;

[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, leaves, etc. are usually selected as explants. Ginseng roots contain more ginsenoside precursors and are ideal materials. For example, select healthy and pest-free ginseng roots and cut them into small pieces, each about 0.5 to 1 cm in length.

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

[0052] 2. Callus induction

[0053] (1) Medium preparation: The commonly used medium for callus induction is MS medium (Murashige and Skoog medium). Appropriate plant growth regulators are added to the 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 medium must contain sucrose as a carbon source at a concentration of about 30 g / L, and agar powder to solidify the medium at a concentration of 7 to 8 g / L.

[0054] (2) Culture conditions: The sterilized explants are inoculated onto the induction medium and cultured in a culture room at 22 to 25°C, with a light intensity of 1000 to 2000 lx and a light duration of 12 to 16 hours per day. After about 2 to 3 weeks, callus tissue begins to form on the surface of the explants, which 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 needed to produce ginsenoside RK3, the induced callus can be transferred to a liquid culture medium for suspension culture. The composition of the liquid culture medium is similar to that of the solid culture 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 culture medium, with an inoculum amount of about 3 to 5 g per bottle. The culture is carried out on a shaker, and the shaker speed is generally 100 to 120 r / min. The temperature and light conditions are the same as those in 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 once 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 Up to 1×10 7 When the cells are 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 suspended culture cells. For example, methyl jasmonate (MeJA) is a commonly used inducer with a concentration of about 100 to 200 μmol / L. It can activate secondary metabolic pathways in cells and promote the synthesis of rare ginsenoside RK3.

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

[0061] 5. Extraction and separation

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

[0063] (2) Separation and purification: The extracted solution is filtered or centrifuged (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. Then, column chromatography, such as silica gel column chromatography, can be used for separation and purification. The crude extract is loaded onto a silica gel column and gradient eluted with a mixed solvent of chloroform-methanol in different proportions. According to the polarity and other properties of the rare ginsenoside RK3, the eluate containing the target compound is collected, and then further purified and identified by methods such as high performance liquid chromatography (HPLC), and finally a high-purity rare ginsenoside RK3 is obtained.

[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 fight fatigue, and the subject can be a mammal, such as a human, rat, rabbit, sheep, pig, cow, cat, dog, monkey, etc.

[0066] The present invention is further described below in conjunction with 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, respectively.

[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 respectively 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.

[0069] Anti-fatigue experiments were conducted on mice using rare ginsenoside RK3, taurine, and compositions 1 to 13. The daily intake of the above compositions for an adult (60 kg) is 1.2 g / 60 kg BW, where BW (bodyweight) stands for (body weight), for example mg / kg BW means how many milligrams of the drug are given 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. The Kunming mice were observed for 3 days before the experiment and all had normal activities.

[0071] Dosage selection: The experimental group was set at 200 mg / kg BW, which was 10 times the daily intake of adults. There were 16 groups in total, namely rare ginsenoside RK3 group, taurine group, combination groups 1 to 13, and blank control group. There were 10 mice in each group. 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 gavage was performed once a day for 30 consecutive days.

[0072] Exhaustive swimming test: Different drug treatment groups were gavaged for 30 days, once a day. Before the experiment, mice were fasted for 4 hours (under fasting conditions, fatigue-related indicators in acute exercise can reflect the true physiological regulation ability). The tails of mice were loaded with a lead wire weighing 5% of their body weight and placed in a 40cm deep swimming pool (65cm high * 20cm diameter) at a water temperature of 34℃±1℃ for forced swimming. The swimming time of the mice was recorded. If their nostrils could not float to the surface within 10s, the swimming time at this time was the exhaustive swimming time of the mice. The exhaustive swimming time of mice in 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 that rare ginsenoside RK3 and taurine are not present, and the control group is used as the basis and is not compared with itself, so p 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 to mice in each test group, an exhaustive swimming test was performed. Compared with the control group, the swimming time of the drug-treated groups increased significantly (p<0.05), compositions 4 to 8 increased extremely significantly compared with the control group (p<0.001), compositions 2 to 9 were significantly different from the rare ginsenoside RK3 and taurine alone (p<0.05), and composition 7 was extremely different from 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 obvious difference in compositions 11 to 13, indicating that they were outside the scope of the present invention and had no synergistic effect.

[0078] Example 2: Effects of rare ginsenoside RK3, taurine, and the combination of rare ginsenoside RK3 and taurine on various serum indices 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] The effects of rare ginsenoside RK3, taurine, and compositions 1 to 13 on various serum indicators of mice after exercise were tested. The daily intake of the above compositions for an adult (60 kg) is 1.2 g / 60 kg BW, where BW (bodyweight) stands for (body weight), for example, mg / kg BW means how many milligrams of the drug are given 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. The Kunming mice were observed for 3 days before the experiment and all had normal activities.

[0082] Dosage selection: The experimental group was set at 200 mg / kg BW, which was 10 times the daily intake of adults. There were 16 groups in total, namely rare ginsenoside RK3 group, taurine group, combination groups 1 to 13, and blank control group. There were 10 mice in each group. 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 gavage was performed once a day for 30 consecutive days.

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

[0084] Different drug treatment groups were gavaged continuously for 30 days, once a day. 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 tails of mice were loaded with a lead wire that was 5% of their body weight and placed in a 40cm deep swimming pool (65cm high * 20cm diameter) at a water temperature of 34℃±1℃ for forced swimming. During forced swimming, mice rested for 60 minutes, and their eyeballs were removed to collect about 1.0mL of whole blood (without anticoagulant) and placed in a 4℃ refrigerator for about 60 minutes. After the blood coagulated, it was centrifuged at 3000rpm / min for 15 minutes, and serum was taken for use. The content of urea nitrogen in the serum was determined using the kit of Boyan Biology, as shown in 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 that rare ginsenoside RK3 and taurine are not present, and the control group is used as the basis and is not compared with itself, so p 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 to mice in each test group, an exhaustive swimming test was performed. 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 obvious (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 obvious 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 compared with the rare ginsenoside RK3 group (p<0.01). Compared with the taurine group alone, the effect of composition 2 to 10 on reducing the serum urea nitrogen of mice was more obvious 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 compared with the taurine group (p<0.01). It shows 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 there is no obvious difference between compositions 11 to 13, which means 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 in the serum of mice.

[0090] (2) Determination of blood lactate content

[0091] Different drug treatment groups were gavaged for 28 consecutive days, once a day. 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 tails of mice were loaded with 5% body weight lead wires and placed in a 40cm deep swimming pool (65cm high * 20cm diameter) at a water temperature of 34℃±1℃ for forced swimming. During forced swimming, mice rested for 60 minutes, and their eyeballs were removed to collect about 1.0mL of whole blood (without anticoagulant) and placed in a 4℃ refrigerator for about 60 minutes. After the blood coagulated, it was centrifuged at 3000rpm / min for 15 minutes, and serum was taken for use. The blood lactate content was measured using Boyan Bio's kit.

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

[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 that rare ginsenoside RK3 and taurine are not present, and the control group is used as the basis and is not compared with itself, so p 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 serum of mice. Compared with the rare ginsenoside RK3 group, the effect of compositions 2 to 10 on reducing serum lactic acid in mice was more obvious (p<0.05) than that of the rare ginsenoside RK3 group, among which composition 7 had a stronger ability to reduce serum lactic acid in mice compared with the rare ginsenoside RK3 group (p<0.01). Compared with the taurine group, the effect of compositions 2 to 10 on reducing serum lactic acid in mice was more obvious (p<0.05) than that of the taurine group, among which composition 7 had a stronger ability to reduce serum lactic acid in mice compared with the taurine group (p<0.01). It shows 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 there is no obvious difference between compositions 11 to 13, indicating that outside the scope of the present invention, the composition has no obvious synergistic effect, but it still has the effect of reducing the serum lactic acid of mice.

[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] The effects of rare ginsenoside RK3, taurine, and compositions 1 to 13 on various serum indicators of mice after exercise were tested. The daily intake of the above compositions for an adult (60 kg) is 1.2 g / 60 kg BW, where BW (bodyweight) stands for (body weight), for example, mg / kg BW means how many milligrams of the drug are given 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. The Kunming mice were observed for 3 days before the experiment and all had normal activities.

[0102] Dosage selection: The experimental group was set at 200 mg / kg BW, which was 10 times the daily intake of adults. There were 16 groups in total, namely rare ginsenoside RK3 group, taurine group, combination groups 1 to 13, and blank control group. There were 10 mice in each group. 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 gavage was performed once a day for 30 consecutive days.

[0103] Different drug treatment groups were gavaged for 28 consecutive days, once a day. 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), and 5% of their body weight was attached to the tail of the mice. They were placed in a 40 cm deep swimming pool (65 cm high * 20 cm diameter) at a water temperature of 34°C ± 1°C for forced swimming. Forced swimming, the mice rested for 60 minutes, and the mice were killed by dislocating the neck. The liver was taken out, rinsed with physiological saline, and then 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. 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 put into a 10 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 poured out, and the test tube was inverted for 10 minutes before the precipitate was taken. The liver glycogen content was determined by the anthrone method.

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

[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 that rare ginsenoside RK3 and taurine are not present, and the control group is used as the basis and is not compared with itself, so p 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 can be seen from 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 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). It shows 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 there is no obvious difference between compositions 11 to 13 and 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, characterized in that It comprises ginsenoside RK3 and taurine, and the weight ratio of the rare ginsenoside RK3 to the taurine is (0.5 to 2):(0.5 to 3).

2. The pharmaceutical composition according to claim 1, characterized in that The weight ratio of the rare ginsenoside RK3 to the taurine is (1 to 1.5):(1 to 2.5).

3. The pharmaceutical composition according to claim 2, characterized in that The weight ratio of the rare ginsenoside RK3 to the taurine is 1.5:(2 to 3).

4. The pharmaceutical composition according to claim 3, characterized in that The weight ratio of the rare ginsenoside RK3 to the taurine is 1.5:

2.

5. A method for preparing a pharmaceutical composition, characterized in that: Used for preparing the pharmaceutical composition as claimed in any one of claims 1 to 4, comprising the steps of: combining rare ginsenoside RK3 and taurine.

6. The method for preparing the pharmaceutical composition according to claim 5, characterized in that: The rare ginsenoside RK3 is obtained by the following steps: Taking ginseng slices after cutting, inoculating them on 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.

7. The method for preparing the pharmaceutical composition according to claim 6, 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.

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

9. The method for preparing the pharmaceutical composition according to claim 6, characterized in that: The column chromatography method uses a silica gel column and performs gradient elution with a chloroform-methanol mixed solvent.

10. Use of the pharmaceutical composition according to any one of claims 1 to 4 in the preparation of anti-fatigue drugs.

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