CN112386637A

CN112386637A - Anti-fatigue pharmaceutical composition and preparation method thereof

Info

Publication number: CN112386637A
Application number: CN201910761873.7A
Authority: CN
Inventors: 林志秀; 冼彦芳
Original assignee: Chinese University of Hong Kong CUHK
Current assignee: Chinese University of Hong Kong CUHK
Priority date: 2019-08-19
Filing date: 2019-08-19
Publication date: 2021-02-23
Anticipated expiration: 2039-08-19
Also published as: CN112386637B

Abstract

The anti-fatigue pharmaceutical composition comprises effective components and medically acceptable excipient, wherein the effective components comprise the following raw materials in percentage by weight: 3-9 parts of astragalus membranaceus, 3-9 parts of adenophora tetraphylla and 3-9 parts of prepared rehmannia root. The pharmaceutical composition has antifatigue effect. The results of in vivo pharmacological experiments prove that the medicament can obviously prolong the time of mouse weight bearing swimming, reduce the content of MDA in gastrocnemius and liver tissues, and increase the activities of SOD, CAT and GSH-Px and the content of glycogen in the gastrocnemius and liver tissues; in addition, the expression of Nrf2 and HO-1 related proteins in tissues of gastrocnemius and liver of mice can be regulated. The invention can be used for preparing the medicine for treating fatigue, and experiments prove that the medicine has the advantages of quick symptom improvement and short treatment course.

Description

Anti-fatigue pharmaceutical composition and preparation method thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to an anti-fatigue pharmaceutical composition and a preparation method thereof.

Background

Fatigue (Fatigue) is the inability of the physiological processes of the body to maintain their function at a particular level or the inability of the organs to maintain their predetermined motor strength. With the rapid development of society, the rhythm of life is accelerated continuously, and fatigue is easily caused by long-term tension work, competitive pressure and emotional instability. Nowadays, fatigue has become one of the major factors that endanger human health. If the fatigue of the body cannot be eliminated in time, the body fatigue will gradually accumulate and cause emotional abnormality, and chronic fatigue syndrome is formed over time, even lead to death due to overstrain. Therefore, the research on the functional activity products with safety, effectiveness and strong universality has very important practical significance.

Fatigue is the inability of the body to maintain its function at a particular level continuously during physiological processes or the inability of organs to maintain their predetermined motor strength. Fatigue is caused by excessive exercise, and long-term cumulative physical fatigue can lead to a series of fatigue-related syndromes, including neurological dysfunction, cognitive problems, sleep disorders, and gastrointestinal symptoms. Nowadays, fatigue has become one of the major factors that endanger human health. Chronic fatigue can have a significant adverse effect on the quality of people's work and life. Although the case of chronic fatigue is ubiquitous, the cause of fatigue is not well understood. At present, chronic fatigue is researched, and the pathogenesis of fatigue is emphasized to be closely related to dysfunction of energy metabolism, immune and endocrine systems, inflammatory response and antioxidant defense systems. Among these potential factors, oxidative stress is the most important cause of fatigue. Oxidative stress increases the production of pro-oxidants, and excessive secretion of Reactive Oxygen Species (ROS) and lipid peroxidation (MDA) can be observed in patients with chronic fatigue syndrome. In chronic fatigue patients, oxidative stress increases the release of proinflammatory cytokines such as tumor necrosis release factor alpha (TNF-alpha), interleukin-6 (IL-6) and interleukin-1 beta (IL-1 beta) by increasing the inflammatory response. Therefore, antioxidants are probably the most valuable method of treating fatigue. However, at present, no antioxidant medicine or treatment method can effectively eliminate the fatigue of patients. Therefore, the search for natural compounds with antioxidation is important, and the natural compounds are developed into safe, effective and universal anti-fatigue foods or medicines, thereby having very important clinical significance for improving the exercise capacity, delaying fatigue and accelerating the elimination of the fatigue state of inpatients.

The radix astragali is dried root of Astragalus membranaceus (Fisch.) Bge. of Leguminosae family or Astragalus membranaceus (Fisch.) Bge. of Hsiao. The herb is recorded in Shen nong Ben Cao Jing (Shen nong's herbal), is a common Chinese herb in folk China, and is also named as Huang Qi, Fa Qi, etc. 2015, recorded in Chinese pharmacopoeia, Astragalus root, radix astragali, sweet in taste, slightly warm in nature, enters lung and spleen meridians. Has the effects of tonifying qi and raising yang, consolidating superficial resistance and arresting sweating, inducing diuresis and relieving swelling, promoting the production of body fluid and nourishing blood, activating stagnancy and removing obstruction of qi, expelling toxin and discharging pus, and astringing sores and promoting granulation, and is used for treating symptoms such as qi deficiency and hypodynamia, anorexia and loose stool, sinking of middle-jiao energy, chronic diarrhea and rectocele, hematochezia and metrorrhagia, superficial deficiency and spontaneous perspiration, qi deficiency and edema, internal heat and thirst, blood deficiency and sallow complexion, hemiplegia, numbness and numbness of pain, carbuncle, cellulitis and difficult ulcer, chronic ulcer and no astringency and the. Modern scientific research finds that astragalus has the function of regulating the immunologic function. The radix astragali extract has various biological activities, such as regulating immunity, promoting hemopoiesis, regulating blood sugar, reducing blood lipid, relieving cerebral ischemia, protecting liver, and resisting tumor.

The radix rehmanniae Preparata is prepared from dried root tuber of Rehmannia glutinosa Libosch of Scrophulariaceae by processing, steaming and drying. The medicine is recorded in Ben Cao Tu Jing (the drawing of materia Medica) and is a traditional Chinese medicine commonly used in folks in China. According to the record of "Chinese pharmacopoeia" in 2015, Shudihuang is sweet in flavor and slightly warm in nature, and enters liver and kidney meridians. Has effects in replenishing blood, nourishing yin, replenishing essence, and replenishing marrow. Can be used for treating blood deficiency, sallow complexion, cardiopalmus, menoxenia, metrorrhagia, metrostaxis, deficiency-type liver-yin and kidney-yin, soreness of waist and knees, hectic fever due to yin deficiency, night sweat, nocturnal emission, internal heat, diabetes, giddiness, tinnitus, and premature gray hair. Modern scientific researches find that the prepared rehmannia root can enhance the immunity of organisms, has various pharmacological effects of resisting oxidation, inhibiting the development of tumors, promoting hematopoiesis, resisting aging and the like, and has no report on the research on the fatigue resistance of the prepared rehmannia root extract at home and abroad.

The Adenophora tetraphylla is dried root of Adenophora tetaphylla (Thunb.) Fisch or Adenophora stricta Miq. It is the traditional Chinese medicine commonly used in folks of China, beginning from Shen nong Ben Cao Jing. 2015 recorded in Chinese pharmacopoeia, the south adenophora root is sweet in taste and slightly cold in nature, and enters lung and stomach meridians. Has the effects of nourishing yin, clearing away lung-heat, benefiting stomach, promoting the production of body fluid, reducing phlegm and tonifying qi. Can be used for treating cough due to lung heat and dryness, cough due to yin deficiency, cough with sticky phlegm, deficiency of stomach yin, anorexia, emesis, deficiency of qi and yin, dysphoria, and xerostomia. Modern scientific researches find that the adenophora tetraphylla has various pharmacological actions of immunoregulation, radiation resistance, aging resistance, free radical elimination, liver protection and the like, and the research on the anti-fatigue of the adenophora tetraphylla extract is not reported at home and abroad.

At present, no document discloses that the composition of astragalus, prepared rehmannia root and adenophora tetraphylla has the anti-fatigue effect and the application thereof in anti-fatigue medicines.

Disclosure of Invention

The invention mainly aims to overcome the defects of the prior art and provide a good and efficient anti-fatigue pharmaceutical composition and a preparation method thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

an anti-fatigue pharmaceutical composition comprises effective components and medically acceptable excipient, wherein the effective components comprise the following raw materials in percentage by weight:

3-9 parts of astragalus membranaceus, 3-9 parts of adenophora tetraphylla and 3-9 parts of prepared rehmannia root.

Further, the effective components comprise the following raw materials in percentage by weight: 6 parts of astragalus membranaceus, 6 parts of adenophora tetraphylla and 6 parts of prepared rehmannia root.

Further, the medicine is granules, capsules, concentrated pills or tablets.

The application of the pharmaceutical composition in preparing the medicine with the anti-fatigue effect.

The preparation method of the anti-fatigue pharmaceutical composition uses effective components and medically acceptable excipient to prepare the pharmaceutical composition, wherein the effective components comprise the following raw materials in percentage by weight:

Further, the preparation method of the pharmaceutical composition comprises the following steps:

(1) weighing the raw materials according to the proportion, adding ethanol, soaking at normal temperature, performing ultrasonic extraction, filtering, and repeating the extraction steps twice;

(2) mixing the three filtrates, recovering under reduced pressure, and freeze drying to obtain extract fine powder;

(3) adding pharmaceutically acceptable excipient, and making into granule, capsule, concentrated pill or tablet by conventional method.

Further, ethanol with the mass percentage concentration of 60% is added in the step (1), the mixture is soaked for 24 hours at normal temperature, and ultrasonic extraction is carried out for 1 hour.

Further, one or more of lactose, dextrin, starch, magnesium stearate, a binder, a solubilizer, a flavoring agent, a bacteriostatic agent, an antioxidant and a coloring agent are added in the step (3).

The invention has the beneficial effects that:

the invention provides an anti-fatigue pharmaceutical composition, which comprises the effective components of astragalus, prepared rehmannia root and adenophora tetraphylla, and has the anti-fatigue effect. The results of in vivo pharmacological experiments prove that the medicament can obviously prolong the time of mouse weight bearing swimming, reduce the content of MDA in gastrocnemius and liver tissues, and increase the activities of SOD, CAT and GSH-Px and the content of glycogen in the gastrocnemius and liver tissues; in addition, the expression of Nrf2 and HO-1 related proteins in tissues of gastrocnemius and liver of mice can be regulated, which is specifically shown as follows: inhibit the activation of proteins in Nrf2/HO-1 pathway, further eliminate oxidative stress, reduce the content of lactic acid, urea sodium and triglyceride in serum, and increase the blood sugar concentration, thereby prolonging the time of mouse weight swimming.

The invention has the advantages that: (1) the pharmaceutical composition has remarkable anti-fatigue biological activity, obviously prolongs the time of mouse weight-bearing swimming after gastric lavage administration, obviously inhibits oxidative stress of gastrocnemius muscle and liver tissue, regulates Nrf2/HO-1 signal path, further influences the metabolism of amino acid and protein of liver tissue, and prolongs the time of mouse weight-bearing swimming. The research result opens up a new application field of the medicine in the aspect of treating the fatigue. (2) The astragalus, the adenophora tetraphylla and the prepared rehmannia root in the active ingredients of the pharmaceutical composition are all derived from traditional and commonly used Chinese medicinal materials in China, can obviously prolong the time of the mice for swimming under load, and can be used for preparing the medicines for treating fatigue. (3) The drug effect substance basis and the biological action mechanism provided by the drug composition provide a new choice for the treatment of fatigue.

Drawings

Figure 1 effect of the pharmaceutical composition of the invention on body weight, food and water intake of mice.

Figure 2 effect of the pharmaceutical composition of the present invention on the weight bearing swimming time of mice (n-12). P <0.05, p <0.01 and p <0.001 were compared to the control group.

Figure 3 effect of the pharmaceutical composition of the invention on oxidative stress in gastrocnemius muscle and liver tissues in swimming-bearing mice (n-6). P <0.05, p <0.01 and p <0.001 were compared to the control group.

Figure 4 effect of the pharmaceutical composition of the invention on adenosine triphosphate and glycogen in gastrocnemius muscle and liver tissues in swimming-bearing mice (n-6).^*p<0.05,^**p<0.01 and^***p<0.001 compared to the control group.

Figure 5 effect of the pharmaceutical composition of the invention on blood lactate, urea sodium, triglycerides and blood glucose in swimming-weight mice (n-6).^*p<0.05,^**p<0.01 and^***p<0.001 compared to the control group.

Figure 6 effect of the pharmaceutical composition of the invention on expression of Nrf2 and HO-1 proteins in gastrocnemius and liver tissues in swimming-bearing mice (n-3). P <0.05, p <0.01 and p <0.001 were compared to the control group.

The notation in the figure is:

Control-Control group;

AR-astragalus;

ADR-adenophora tetraphylla;

RRP-prepared rehmannia root.

Detailed Description

The embodiments of the present invention will be described in detail below. It should be emphasized that the following description is merely exemplary in nature and is not intended to limit the scope of the invention or its application.

An anti-fatigue pharmaceutical composition comprises effective components and medically acceptable excipient, and is characterized in that the effective components comprise the following raw materials in percentage by weight: 3-9 parts of astragalus membranaceus, 3-9 parts of adenophora tetraphylla and 3-9 parts of prepared rehmannia root.

In a preferred embodiment, the effective components comprise the following raw materials in percentage by weight: 6 parts of astragalus membranaceus, 6 parts of adenophora tetraphylla and 6 parts of prepared rehmannia root.

Optionally, the medicament is granules, capsules, concentrated pills or tablets.

The preparation method of the anti-fatigue pharmaceutical composition uses effective components and medically acceptable excipient to prepare the pharmaceutical composition, wherein the effective components comprise the following raw materials in percentage by weight: 3-9 parts of astragalus membranaceus, 3-9 parts of adenophora tetraphylla and 3-9 parts of prepared rehmannia root.

In a preferred embodiment, the preparation method of the pharmaceutical composition comprises the following steps:

(1) weighing the raw materials according to the proportion, adding 60% ethanol by mass, soaking at normal temperature for 24h, performing ultrasonic extraction for 1h, filtering, and repeating the extraction steps twice;

(3) adding pharmaceutically acceptable excipient such as one or more of lactose, dextrin, starch, magnesium stearate, binder, solubilizer, correctant, antibacterial, antioxidant, and colorant, and making into granule, capsule, concentrated pill or tablet by conventional method.

Specifically, the pharmaceutical composition is prepared from 3-9 parts by weight of astragalus membranaceus, 3-9 parts by weight of adenophora tetraphylla, 3-9 parts by weight of prepared rehmannia root and an optional medically acceptable excipient. Can be made into granule, capsule, concentrated pill or tablet. The experiment proves that the traditional Chinese medicine composition has the advantages of quick symptom improvement and short treatment course. The medicine of the invention has few kinds of raw materials, simple and easy operation of the preparation method and wide application value. After the medicine is orally taken, the time of mouse weight bearing swimming is obviously prolonged, and the blood sugar concentration in serum, the Adenosine Triphosphate (ATP) and the glycogen content in gastrocnemius and liver tissues are obviously improved; inhibiting the levels of lactic acid (lactic acid), Blood Urea Nitrogen (BUN), Triglycerides (Triglycerides) in serum, inhibiting Malondialdehyde (MDA) levels in the gastrocnemius and liver tissues, while increasing the levels of superoxide dismutase (SOD), Catalase (CAT) and glutathione peroxidase (GSH-Px) and inhibiting activation of the Nrf2/HO-1 signaling pathway. The experimental result proves that the medicine can eliminate oxidative stress and effectively regulate the metabolism of protein and amino acid in liver and gastrocnemius tissues by regulating the activation of an Nrf2/HO-1 signal channel under a certain dosage, thereby obviously prolonging the time of mouse weight-bearing swimming and having the new application of the anti-fatigue medicine.

Example 1 (tablet)

1000g of the medicine extract is taken, 480g of lactose and 500g of starch are added and mixed uniformly, 300g of 7% starch slurry is used as an adhesive, wet granulation is carried out, drying is carried out, 20g of magnesium stearate is added and mixed uniformly, 10000 tablets containing 100mg of the extract in each tablet are pressed, and the net weight of each tablet is 0.2 g. It is administered orally 2 times a day, 3 tablets each time, and can be used for relieving fatigue. The symptoms are as follows: tired and not good after rest.

Example 2 (tablet)

Taking 800g of the medicine extract, adding 480g of lactose and 700g of starch, uniformly mixing, using 300g of 7% starch slurry as an adhesive, carrying out wet granulation, drying, adding 20g of magnesium stearate, uniformly mixing, and pressing into 10000 tablets containing 80mg of the extract per tablet, wherein the net weight of each tablet is 0.2 g. It is administered orally 2 times a day, 3 tablets each time, and can be used for relieving fatigue. The symptoms are as follows: tired and not good after rest.

Example 3 (tablet)

Taking 500g of the medicine extract, adding 480g of lactose and 1000g of starch, uniformly mixing, using 300g of 7% starch slurry as an adhesive, carrying out wet granulation, drying, adding 20g of magnesium stearate, uniformly mixing, and pressing into 10000 tablets containing 50mg of the extract per tablet, wherein the net weight of each tablet is 0.2 g. It is administered orally 2 times a day, 3 tablets each time, and can be used for relieving fatigue. The symptoms are as follows: fatigue of the whole body, failure to improve after rest, etc.

Example 4 (tablet)

1000g of the medicine extract is taken, 2984g of starch is added and mixed uniformly, 350g of 7% starch slurry is used as an adhesive, wet granulation is carried out, drying is carried out, 16g of magnesium stearate is added and mixed uniformly, 10000 tablets containing 100mg of extract in each tablet are pressed, and the net weight of each tablet is 0.4 g. It is administered orally 2 times a day, 3 tablets each time, and can be used for relieving fatigue. The symptoms are as follows: fatigue of the whole body, failure to improve after rest, etc.

Example 5 (tablet)

Taking 800g of the medicine extract, adding 4016g of starch, mixing uniformly, using 350g of 7% starch slurry as an adhesive, granulating by a wet method, drying, adding 16g of magnesium stearate, mixing uniformly, and pressing into 10000 tablets containing 80mg of the extract per tablet, wherein the net weight of each tablet is 0.4 g. It is administered orally 2 times a day, 3 tablets each time, and can be used for relieving fatigue. The symptoms are as follows: fatigue of the whole body, failure to improve after rest, etc.

Example 6 (tablet)

500g of the medicine extract is taken, 3484g of starch is added and mixed evenly, 350g of 7% starch slurry is used as an adhesive, wet granulation is carried out, drying is carried out, 16g of magnesium stearate is added and mixed evenly, and 10000 tablets containing 50mg of the extract in each tablet are pressed, and the net weight of each tablet is 0.4 g. It is administered orally 2 times a day, 3 tablets each time, and can be used for relieving fatigue. The symptoms are as follows: fatigue of the whole body, failure to improve after rest, etc.

Example 7 (tablet)

1000g of the medicine extract is taken, 594g of lactose and 390g of starch are added and mixed uniformly, 350g of 7% starch slurry is used as an adhesive, wet granulation is carried out, drying is carried out, 16g of magnesium stearate is added and mixed uniformly, 10000 tablets containing 100mg of extract per tablet are pressed, and the net weight of each tablet is 0.2 g. It is administered orally 2 times a day, 3 tablets each time, and can be used for relieving fatigue. The symptoms are as follows: fatigue of the whole body, failure to improve after rest, etc.

Example 8 (tablet)

Taking 800g of the medicine extract, adding 614g of lactose, uniformly mixing 570g of starch, using 350g of 7% starch slurry as an adhesive, granulating by a wet method, drying, adding 16g of magnesium stearate, uniformly mixing, and pressing into 10000 tablets containing 80mg of the extract per tablet, wherein the net weight of each tablet is 0.2 g. It is administered orally 2 times a day, 3 tablets each time, and can be used for relieving fatigue. The symptoms are as follows: fatigue of the whole body, failure to improve after rest, etc.

Example 9 (tablet)

Taking 500g of the medicine extract, adding 574g of lactose, uniformly mixing, using 350g of 7% starch slurry as an adhesive, carrying out wet granulation, drying, adding 16g of magnesium stearate, uniformly mixing, and pressing into 10000 tablets containing 50mg of the extract per tablet, wherein the net weight of each tablet is 0.2 g. It is administered orally 2 times a day, 3 tablets each time, and can be used for relieving fatigue. The symptoms are as follows: fatigue of the whole body, failure to improve after rest, etc.

Example 10 (tablet)

1000g of the medicine extract is taken, 480g of dextrin and 1114g of starch are added and mixed uniformly, 350g of 7% starch slurry is used as an adhesive, wet granulation is carried out, drying is carried out, 16g of magnesium stearate is added and mixed uniformly, 10000 tablets containing 100mg of the extract in each tablet are pressed, and the net weight of each tablet is 0.1 g. It is administered orally 2 times a day, 3 tablets each time, and can be used for relieving fatigue. The symptoms are as follows: fatigue of the whole body, failure to improve after rest, etc.

Example 11 (tablet)

Taking 800g of the medicine extract, adding 480g of dextrin and 1134g of starch, uniformly mixing, using 350g of 7% starch slurry as an adhesive, granulating by a wet method, drying, adding 16g of magnesium stearate, uniformly mixing, and pressing into 10000 tablets containing 80mg of the extract per tablet, wherein the net weight of each tablet is 0.2 g. It is administered orally 2 times a day, 3 tablets each time, and can be used for relieving fatigue. The symptoms are as follows: fatigue of the whole body, failure to improve after rest, etc.

Example 12 (tablet)

Taking 500g of the medicine extract, adding 480g of dextrin and 1094g of starch, uniformly mixing, using 350g of 7% starch slurry as an adhesive, granulating by a wet method, drying, adding 16g of magnesium stearate, uniformly mixing, and pressing into 10000 tablets containing 50mg of the extract per tablet, wherein the net weight of each tablet is 0.2 g. It is administered orally 2 times a day, 3 tablets each time, and can be used for relieving fatigue. The symptoms are as follows: fatigue of the whole body, failure to improve after rest, etc.

Example 13 (tablet)

1000g of the medicine extract is taken, 480g of lactose and 1114g of starch are added and mixed uniformly, 350g of 7% starch slurry is used as an adhesive, wet granulation is carried out, drying is carried out, 16g of talcum powder is added and mixed uniformly, 10000 tablets containing 100mg of the extract are pressed into each tablet, and the net weight of each tablet is 0.2 g. It is administered orally 2 times a day, 3 tablets each time, and can be used for relieving fatigue. The symptoms are as follows: fatigue of the whole body, failure to improve after rest, etc.

Example 14 (tablet)

Taking 800g of the medicine extract, adding 480g of lactose and 1134g of starch, uniformly mixing, using 350g of 7% starch slurry as an adhesive, carrying out wet granulation, drying, adding 16g of talcum powder, uniformly mixing, and pressing into 10000 tablets containing 80mg of the extract per tablet, wherein the net weight of each tablet is 0.2 g. It is administered orally 2 times a day, 3 tablets each time, and can be used for relieving fatigue. The symptoms are as follows: fatigue of the whole body, failure to improve after rest, etc.

Example 15 (tablet)

Taking 500g of the medicine extract, adding 480g of lactose and 1094g of starch, uniformly mixing, using 350g of 7% starch slurry as an adhesive, carrying out wet granulation, drying, adding 16g of talcum powder, uniformly mixing, and pressing into 10000 tablets containing 50mg of the extract per tablet, wherein the net weight of each tablet is 0.2 g. It is administered orally 2 times a day, 3 tablets each time, and can be used for relieving fatigue. The symptoms are as follows: fatigue of the whole body, failure to improve after rest, etc.

Example 16 (tablet)

1000g of the medicine extract is taken, 480g of dextrin and 504g of starch are added and mixed uniformly, 350g of 7% starch slurry is used as an adhesive, wet granulation is carried out, drying is carried out, 16g of talcum powder is added and mixed uniformly, 10000 tablets containing 100mg of the extract are pressed into each tablet, and the net weight of each tablet is 0.2 g. It is administered orally 2 times a day, 3 tablets each time, and can be used for relieving fatigue. The symptoms are as follows: fatigue of the whole body, failure to improve after rest, etc.

Example 17 (tablet)

Taking 800g of the medicine extract, adding 480g of dextrin and 704g of starch, uniformly mixing, using 350g of 7% starch slurry as an adhesive, carrying out wet granulation, drying, adding 16g of talcum powder, uniformly mixing, and pressing into 10000 tablets containing 80mg of the extract per tablet, wherein the net weight of each tablet is 0.2 g. It is administered orally 2 times a day, 3 tablets each time, and can be used for relieving fatigue. The symptoms are as follows: fatigue of the whole body, failure to improve after rest, etc.

Example 18 (tablet)

500g of the medicine extract is taken, 480g of dextrin and 1004g of starch are added and mixed uniformly, 350g of 7% starch slurry is used as an adhesive, wet granulation is carried out, drying is carried out, 16g of talcum powder is added and mixed uniformly, 10000 tablets containing 50mg of the extract are pressed into each tablet, and the net weight of each tablet is 0.2 g. It is administered orally 2 times a day, 3 tablets each time, and can be used for relieving fatigue. The symptoms are as follows: fatigue of the whole body, failure to improve after rest, etc.

Example 19 (capsules)

1000g of the medicine extract is taken, 980g of lactose and 1004g of starch are added and mixed uniformly, 350g of 7% starch slurry is used as an adhesive, wet granulation is carried out, drying is carried out, 16g of magnesium stearate is added and mixed uniformly, and the mixture is filled into No. 1 capsules to be made into 10000 capsules, wherein 100mg of the extract is contained in each capsule, and the net weight of each capsule is 0.3 g. It is administered orally 2 times a day, 3 granules each time, and can be used for relieving fatigue. The symptoms are as follows: fatigue of the whole body, failure to improve after rest, etc.

Example 20 (capsules)

Taking 800g of the medicine extract, adding 980g of lactose and 1204g of starch, uniformly mixing, using 350g of 7% starch slurry as an adhesive, carrying out wet granulation, drying, adding 16g of magnesium stearate, uniformly mixing, filling into No. 1 capsules, and preparing into 10000 capsules, wherein each capsule contains 80mg of the extract, and the net weight of each tablet is 0.3 g. It is administered orally, 2 times daily, 3 granules each time, and can be used for relieving fatigue. The symptoms are as follows: fatigue of the whole body, failure to improve after rest, etc.

Example 21 (capsules)

Taking 500g of the medicine extract, adding 980g of lactose and 1504g of starch, mixing uniformly, using 350g of 7% starch slurry as an adhesive, granulating by a wet method, drying, adding 16g of magnesium stearate, mixing uniformly, filling into No. 1 capsules to prepare 10000 capsules, wherein each capsule contains 50mg of the extract, and the net weight of each tablet is 0.3 g. It is administered orally 2 times a day, 3 granules each time, and can be used for relieving fatigue. The symptoms are as follows: fatigue of the whole body, failure to improve after rest, etc.

Example 22 (capsules)

1000g of the medicine extract is taken, 2984g of starch is added and mixed uniformly, 350g of 7% starch slurry is used as an adhesive, wet granulation is carried out, drying is carried out, 16g of magnesium stearate is added and mixed uniformly, and the mixture is filled into No. 1 capsules to be prepared into 10000 capsules, wherein the net weight of each capsule is 0.4g, and each capsule contains 100mg of the extract. It is administered orally 2 times a day, 3 granules each time, and can be used for relieving fatigue. The symptoms are as follows: fatigue of the whole body, failure to improve after rest, etc.

Example 23 (capsules)

Taking 800g of the medicine extract, adding 3184g of starch, uniformly mixing, using 350g of 7% starch slurry as an adhesive, granulating by a wet method, drying, adding 16g of magnesium stearate, uniformly mixing, filling into No. 1 capsules, preparing into 10000 capsules, wherein each capsule contains 80mg of the extract, and the net weight of each capsule is 0.4 g. It is administered orally 2 times a day, 3 granules each time, and can be used for relieving fatigue. The symptoms are as follows: fatigue of the whole body, failure to improve after rest, etc.

Example 24 (capsules)

500g of the medicine extract is taken, 3484g of starch is added and mixed uniformly, 350g of 7% starch slurry is used as an adhesive, wet granulation is carried out, drying is carried out, 16g of magnesium stearate is added and mixed uniformly, and the mixture is filled into No. 1 capsules to be prepared into 100000 capsules, wherein each capsule contains 50mg of the extract, and the net weight of each capsule is 0.4 g. It is administered orally 2 times a day, 3 granules each time, and can be used for relieving fatigue. The symptoms are as follows: fatigue of the whole body, failure to improve after rest, etc.

Example 25 (capsules)

1000g of the medicine extract is taken, 1480g of lactose and 2504g of starch are added and mixed uniformly, 350g of 7% starch slurry is used as an adhesive, wet granulation is carried out, drying is carried out, 16g of magnesium stearate is added and mixed uniformly, and the mixture is filled into No. 0 capsules to be made into 10000 capsules, wherein each capsule contains 100mg of the extract, and the net weight of each tablet is 0.5 g. It is administered orally 2 times a day, 3 granules each time, and can be used for relieving fatigue. The symptoms are as follows: fatigue of the whole body, failure to improve after rest, etc.

Example 26 (capsules)

Taking 800g of the medicine extract, adding 1480g of lactose and 2704g of starch, uniformly mixing, using 350g of 7% starch slurry as an adhesive, granulating by a wet method, drying, adding 16g of magnesium stearate, uniformly mixing, filling into No. 0 capsules, and preparing into 10000 capsules, wherein each capsule contains 80mg of the extract, and the net weight of each capsule is 0.5 g. It is administered orally 2 times a day, 3 granules each time, and can be used for relieving fatigue. The symptoms are as follows: fatigue of the whole body, failure to improve after rest, etc.

Example 27 (capsules)

Taking 600g of the medicine extract, adding 1480g of lactose and 2904g of starch, uniformly mixing, using 350g of 7% starch slurry as an adhesive, granulating by a wet method, drying, adding 16g of magnesium stearate, uniformly mixing, filling into No. 0 capsules, and preparing into 10000 capsules, wherein each capsule contains 50mg of the extract, and the net weight of each capsule is 0.5 g. It is administered orally 2 times a day, 3 granules each time, and can be used for relieving fatigue. The symptoms are as follows: fatigue of the whole body, failure to improve after rest, etc.

Example 28 (capsules)

500g of the medicine extract is taken, 1480g of lactose and 3004g of starch are added and mixed uniformly, 350g of 7% starch slurry is used as an adhesive, wet granulation is carried out, drying is carried out, 16g of magnesium stearate is added and mixed uniformly, and the mixture is filled into No. 2 capsules to be 10000 capsules, wherein each capsule contains 50mg of the extract, and the net weight of each capsule is 0.2 g. It is administered orally 2 times a day, 3 granules each time, and can be used for relieving fatigue. The symptoms are as follows: fatigue of the whole body, failure to improve after rest, etc.

Example 29 (capsules)

400g of the medicine extract is taken, 480g of lactose and 1104g of starch are added and mixed uniformly, 350g of 7% starch slurry is used as an adhesive, wet granulation is carried out, drying is carried out, 16g of magnesium stearate is added and mixed uniformly, and the mixture is filled into No. 2 capsules to be 10000 capsules, wherein each capsule contains 40mg of the extract, and the net weight of each capsule is 0.2 g. It is administered orally 2 times a day, 3 granules each time, and can be used for relieving fatigue. The symptoms are as follows: fatigue of the whole body, failure to improve after rest, etc.

Example 30 (capsules)

250g of the medicine extract is taken, 580g of lactose and 1154g of starch are added and mixed uniformly, 350g of 7% starch slurry is used as an adhesive, wet granulation is carried out, drying is carried out, 16g of magnesium stearate is added and mixed uniformly, and the mixture is filled into No. 2 capsules to be made into 10000 capsules, wherein each capsule contains 25mg of the extract, and the net weight of each capsule is 0.2 g. It is administered orally 2 times a day, 3 granules each time, and can be used for relieving fatigue. The symptoms are as follows: fatigue of the whole body, failure to improve after rest, etc.

Example 31 (oral liquid)

Taking 50g of the medicine extract, adding a solubilizer, namely the poloxamer and a certain proportion of ethanol, and fully stirring uniformly; adding appropriate additives (such as correctant, antibacterial agent, antioxidant, and colorant), dissolving, filtering, clarifying, packaging into ampoule or pop-top bottle, and sterilizing. The specification is that each bottle contains 166mg of the extract oral liquid, and the net content of each bottle is 10 mL. It is administered orally 1 bottle 1 time per day for relieving fatigue. The symptoms are as follows: fatigue of the whole body, failure to improve after rest, etc.

Example 32 (oral liquid)

Taking 100g of the medicine extract, adding ethanol in a certain proportion, and fully stirring; adding appropriate additives (such as correctant, antibacterial agent, antioxidant, and colorant), dissolving, filtering, clarifying, packaging into ampoule or pop-top bottle, and sterilizing. The specification is that each bottle contains 300mg of the extract oral liquid, and the net content of each bottle is 10 mL. It is administered orally 1 bottle 1 time per day for relieving fatigue. The symptoms are as follows: fatigue of the whole body, failure to improve after rest, etc.

Example 33 (oral liquid)

Taking 200g of the medicine extract, adding ethanol in a certain proportion, and fully stirring; adding appropriate additives (such as correctant, antibacterial agent, antioxidant, and colorant), dissolving, filtering, clarifying, packaging into ampoule or pop-top bottle, and sterilizing. The specification is that each bottle contains 600mg of the extract oral liquid, and the net content of each bottle is 10 mL. It is administered orally 1 bottle 1 time per day for relieving fatigue. The symptoms are as follows: fatigue of the whole body, failure to improve after rest, etc.

Example 34 (oral liquid)

Taking 300g of the medicine extract, adding ethanol in a certain proportion, and fully stirring; adding appropriate additives (such as correctant, antibacterial agent, antioxidant, and colorant), dissolving, filtering, clarifying, packaging into ampoule or pop-top bottle, and sterilizing. The specification is that each bottle contains 900mg of the extract oral liquid, and the net content of each bottle is 10 mL. It is administered orally 1 bottle 1 time per day for relieving fatigue. The symptoms are as follows: fatigue of the whole body, failure to improve after rest, etc.

Example 35 (oral liquid)

Taking 400g of the medicine extract, adding ethanol in a certain proportion, and fully stirring; adding appropriate additives (such as correctant, antibacterial agent, antioxidant, and colorant), dissolving, filtering, clarifying, packaging into ampoule or pop-top bottle, and sterilizing. The specification is that each bottle contains 1333mg of extract oral liquid, and the net content of each bottle is 10 mL. It is administered orally 1 bottle 1 time per day for relieving fatigue. The symptoms are as follows: fatigue of the whole body, failure to improve after rest, etc.

EXAMPLE 36 (oral liquid)

Taking 500g of the medicine extract, adding ethanol in a certain proportion, and fully stirring; adding appropriate additives (such as correctant, antibacterial agent, antioxidant, and colorant), dissolving, filtering, clarifying, packaging into ampoule or pop-top bottle, and sterilizing. The specification is that each bottle of oral liquid contains 1666mg of extract, and the net content of each bottle is 10 mL. It is administered orally 1 bottle 1 time per day for relieving fatigue. The symptoms are as follows: fatigue of the whole body, failure to improve after rest, etc.

The drug of the invention is used for observing the drug effect of the drug on the mouse anti-fatigue through in-vivo mouse weight-bearing swimming experiments, and analyzing the possible action mechanism of the drug. Through mouse weight swimming experiments, the composition can obviously prolong the weight swimming time of mice, and the action mechanism of the composition is probably related to inhibiting the activation of the Nrf2/HO-1 pathway, further eliminating oxidative stress, increasing glycogen content and prolonging the weight swimming time of the mice.

The effect is as follows:

1. the inventor proves that the drug can obviously prolong the time of heavy swimming of mice through in vivo experiments. The medicine can well prolong the time of mouse weight swimming, can be used for preparing anti-fatigue medicine, and provides a new choice for the treatment of fatigue.

2. At present, the pathogenesis of fatigue, especially the pathogenesis of cell factors, is more and more emphasized by people. The experiment proves that the medicine can obviously reduce the content of MDA in gastrocnemius and liver tissues, increase the activities of SOD, CAT and GSG-Px in the gastrocnemius and liver tissues, and clarify the anti-fatigue action mechanism from the perspective of oxidative stress. In addition, the medicine can obviously reduce the content of lactic acid, urea nitrogen and triglyceride in serum, improve the blood sugar concentration, increase the content of glycogen and adenosine triphosphate in gastrocnemius muscle and liver tissues, and illustrate the anti-fatigue action mechanism of the medicine from the perspective of regulating the catabolism of amino acid and protein in liver.

Experimental methods

The invention is proved by in vivo experiments that the composition of astragalus, prepared rehmannia root and adenophora tetraphylla can prolong the weight swimming time of mice, and MDA, CAT, SOD, GSH-Px and glycogen content in gastrocnemius muscle and liver tissues and blood sugar, lactic acid, urea nitrogen and triglyceride in serum are measured by adopting a biochemical kit; in addition, the expression difference of Nrf2 and HO-1 proteins in gastrocnemius and liver tissues is measured by an immunoblotting method, and the medicament can be further proved to be capable of obviously prolonging the time of mouse weight-bearing swimming, so that the medicament has a new medical application of fatigue resistance.

1.1 Experimental materials

1.1.1 Experimental animals and drugs

SPF grade C57 mice, male, 22-24g, were purchased from the university of Chinese laboratory animals center, hong Kong. The experimental animals are raised in cages, the padding is replaced every 2 days, adaptive feeding is carried out for 7 days, and drinking water and eating are free during the experiment period. The temperature of the breeding environment is 22 +/-2 ℃, the relative humidity is 60%, and the breeding environment is irradiated for 12 hours every day and circulated for 12 hours in the dark.

1.1.2 preparation of test drugs

The source is as follows: the composition of astragalus root, adenophora tetraphylla and prepared rehmannia root is extracted from a laboratory.

Preparing the extracts of the composition of astragalus, adenophora tetraphylla and prepared rehmannia root: 33.3 percent of astragalus root, 33.3 percent of adenophora tetraphylla and 33.3 percent of prepared rehmannia root, adding 10 times of 60 percent ethanol, soaking for 24 hours, carrying out ultrasonic extraction for 1 hour, filtering, and repeating the extraction steps twice. Combining the three filtrates, recovering under reduced pressure, and freeze drying. The calculated extraction rate was 30.61%. The extracts of the above pharmaceutical compositions were formulated into test solution samples at concentrations of 0.125, 0.25, and 0.5g of the original drug substance per mL.

The preparation method of the combined extract of the astragalus root, the adenophora tetraphylla and the prepared rehmannia root is consistent with the preparation method of the combined extract of the astragalus root, the adenophora tetraphylla and the prepared rehmannia root. The extraction rate is 30.61%

Positive control test solution (AA): precisely weighing 1.3g ascorbic acid dissolved in 130mL distilled water to prepare a sample of the test solution with the concentration of 10 mg/mL.

1.1.3 the medicine is prepared and stored in a refrigerator at 4 ℃ for standby. Experimental reagent

Molding and dosing reagent: ascorbic Acid (AA), Sigma, usa; distilled water, drochen; coomassie brilliant blue protein quantitation reagent, Bio-Rad; mouse lactic acid, BUN, triglycerides, MDA, CAT, SOD, GPx, adenosine triphosphate and glycogen biochemical kit, Abcam, usa.

Western blot determination related reagents: RIPA lysate, protease inhibitor cocktail, PMSF (100mM), phosphorylated protease inhibitor, protein loading buffer, SDS-PAGE gel preparation reagent, Sigma, USA; protein Marker, Thermo Fisher Scientific; TRIS buffer, glycine, SDS, Sigma, usa; PVDF membrane (0.22 μm), Bio-Rad; BSA, Thermo Scientific; tween-20, Sigma, USA; beta-actin, R & D Systems, Histone H3, HRP-labeled goat anti-rabbit, HRP-labeled donkey anti-goat, HRP-labeled goat anti-mouse, HRP-labeled goat anti-rat, Thermo Scientific, Inc.

1.1.4 Experimental instruments

752-P ultraviolet spectrophotometer, Shanghai Shikejie Instrument Co., Ltd; SHIMADZU analytical balance, guangzhou xiang instrument electromechanical devices ltd; PL-203 electronic balance, mettler-toledo instruments (shanghai) ltd; IKA10 tissue homogenizer, IKA, Germany; model BH22 optical microscope, Olympus, japan; FLUO star Optima microplate reader, BMG Labtec, Germany; ABI-7500 fluorescent quantitative PCR instrument, ABI corporation, USA; FBZ2001-up-p Standard reagent type Water purification apparatus, Fulemm technologies, Inc. of Qingdao; bench high speed refrigerated centrifuge, Heal force corporation; DYY-6C electrophoresis apparatus, Beijing Liuyi instruments factory.

1.1.5 animal grouping and administration

Each mouse was weighed before the start of the experiment and randomly grouped by weight: normal group (Control), group (1.25g drug/kg), drug of the invention medium dose group (2.5g drug/kg), drug of the invention group (5.0g drug/kg), ascorbic acid group (AA: 100mg/kg), each group of 12 mice. After grouping, labeling, each mouse was weighed again, and the corresponding dose volume (10mL/kg) was recorded and calculated. The normal group was given the same volume of distilled water. Each group of mice was gavaged with the above extract once a day for 28 days.

1.1.6 weight bearing swimming test of mice

During the experiment, the body weight and activity of the mice were recorded daily and the body weight was recorded every three days. After 1 hour from the last administration, the mice were weighed and the roots of the rat tails were loaded with 10% iron wire. Respectively putting the mice into a swimming box for swimming, wherein the water depth in the box is not less than 30cm, and the water temperature is (20 +/-1.0) DEG C, and recording the time from the beginning of the weight swimming of the mice to the time that the head of the mice is immersed in the water for 5s and cannot return to the water surface as the weight swimming time of the mice.

1.1.790 minute free swimming test

During the experiment, the body weight and activity of the mice were recorded daily and the body weight was recorded every three days. 1 hour after the last dose, the mice underwent a 90 minute freestyle experiment without weight loading. After the experiment is finished, the mice are rested on the bank for 1 hour, then the mice are completely killed by a cervical dislocation method through an orbit blood taking method, and gastrocnemius and liver tissues are immediately dissected.

1.1.8 taking materials from gastrocnemius and liver

After the experiment is finished, the mice are completely killed by a cervical dislocation method through an orbit blood sampling method, gastrocnemius and liver tissues are dissected immediately after blood sampling, and 6 mice are randomly selected from each group and placed in an EP (EP) tube for subsequent glycogen determination; in addition, 6 tissues of gastrocnemius and liver of the mice in each group are randomly taken and placed in an EP (EP) tube for subsequent WB (wideband) determination; the remaining mice, gastrocnemius and liver (6), were placed in additional EP tubes for Elisa and oxidation kit assays. After the materials are collected, the materials are placed into a refrigerator with the temperature of 80 ℃ below zero for storage and are used in subsequent experiments.

1.1.8.1 Biochemical kit for determining oxidative stress change

The tissue weight was accurately determined with reference to the kit instructions, and tissue homogenates were prepared to determine the amounts of MDA, CAT, SOD, GPx, adenosine triphosphate and glycogen in the gastrocnemius and liver tissues of each group.

1.1.9 Western Blot to detect expression of related proteins

1.2.6.1 Total protein extraction

The tissue blocks were washed 2-3 times with cold PBS, decontaminated, cut into small pieces and placed in a homogenizer. The homogenate was thoroughly homogenized on ice by adding 10 tissue volume homogenate (protease inhibitor added within minutes before use). The homogenate was transferred to a 1.5mL centrifuge tube and shaken. And (3) reacting for 30min on ice bath, repeatedly blowing and beating by using a pipette during the reaction to ensure that the cells are completely lysed, centrifuging for 10min at 12000g, and collecting supernatant, namely the total protein solution.

1.2.7.2 cytosolic protein extraction

The tissue is cut into very fine pieces as much as possible. Appropriate amounts of cytosolic protein extraction reagents a and B were mixed at a ratio of 20: 1. And PMSF was added to a final concentration of 1mM to prepare a tissue homogenate. The tissue and tissue homogenate were mixed at a rate of 300 microliters of tissue homogenate per 100mg of tissue and thoroughly homogenized in a glass homogenizer. Homogenization was carried out in an ice bath or at 4 ℃. After homogenization the homogenate was transferred to a plastic centrifuge tube and placed in an ice bath for 30 minutes. Centrifuge at 12000g for 15 min at 4 ℃. The supernatant was transferred to a pre-cooled plastic tube to extract a portion of the resulting cytoplasmic protein.

1.2.7.3 protein concentration assay: coomassie brilliant blue method for measuring protein concentration

1.2.7.4 SDS-PAGE

SDS-PAGE gels were formulated with reference to the SDS-PAGE gel preparation kit instructions. Adding sufficient electrophoresis liquid and loading for electrophoresis. Adding the sample into an electrophoresis hole, and carrying out electrophoresis. The voltage of the concentrated gel is 80V, and the voltage of the separation gel is 110V. And (5) stopping electrophoresis until bromophenol blue just runs out, and performing membrane transfer.

1.2.7.5 transfer film

6 pieces of 7X 9cm filter paper and a piece of moderately sized PVDF membrane were prepared, which was activated with methanol before use. The transfer solution is added to the basin, and the clamp for transferring the membrane, the two sponge pads, the filter paper and the activated PVDF membrane are placed in the basin. The clamp is opened to keep the black side horizontal. The pad is padded with sponge and three layers of filter paper. Carefully peel off the separation gel cover on the filter paper, cover the membrane on the gel, and remove the air bubbles. Three filter papers were covered on the membrane and the air bubbles were removed. Finally, another spongy cushion is covered. Film transfer conditions (wet transfer): fast rotation, and constant rotation of 250mA for 90 minutes; and (5) performing semidry method rotation, and performing membrane rotation for 45min at constant pressure of 20V.

1.2.7.6 immune response

The transferred membranes were destained at room temperature on a shaker and blocked with 5% skim milk (0.5% TBST) for 2 h. Primary antibody (TBST solubilized 5% skim milk, phosphorylated using TBST solubilized 5% BSA) was diluted and incubated overnight at 4 ℃. Washing with TBST on a decolorizing table at room temperature for three times (5 min each), diluting the secondary antibody 3000 times with TBST, incubating at room temperature for 1 hr, and washing with TBST on a decolorizing table at room temperature for three times (5 min each).

1.2.7.7 chemiluminescence

Mixing ECLA and ECLB reagents in equal volume in a centrifuge tube, fully contacting the PVDF membrane with the mixed solution with the protein surface facing upwards, removing residual liquid after 1-2min, wrapping, and exposing in a dark box. And finally, developing and fixing by using a developing and fixing agent. The exposure conditions are adjusted according to the different light intensities.

1.2.8 statistical analysis

The data are expressed as mean ± standard error mean (mean ± SEM), the graph is drawn and statistically analyzed by using Graphpad Prism 5 software, the difference between groups is compared by using One-way analysis of variance (One-WayANOVA), and the difference between groups is significantly different by using Dunnett's method in multiple comparisons of two-by-two, with p <0.05 as a standard. In addition p <0.05, p <0.01 and p <0.001 compared to the Control group.

1.3 results of the experiment

1.3.1 Effect of extracts of the inventive drugs on mouse body weight, grain and water intake

The research shows that the medicament has no influence on the weight, the grain and the water intake of the mouse. The body weight, grain and water intake changes for each group are shown in fig. 1 as a-C.

1.3.2 Effect of the drug of the present invention on the time of swimming with weight of mice

In addition, as shown in A in figure 2, the low, medium and high dose groups of the drug of the invention can obviously prolong the time of heavy swimming of mice after 28 days of administration, which indicates that the drug of the invention has obvious anti-fatigue effect.

1.3.3 Effect of the inventive Agents on oxidative stress in gastrocnemius muscle and liver tissues in swimming-weight bearing mice

As shown in a-H in fig. 3, the drug group of the present invention significantly decreased Malondialdehyde (MDA) levels in gastrocnemius muscle and liver tissues, while the levels of superoxide dismutase (SOD), Catalase (CAT), and glutathione peroxidase (GSH-Px) were significantly increased, as compared to the control group. AA also has a certain antioxidation effect when being administered, but the effect is not as good as that of the medicament of the invention.

1.3.4 Effect of the drugs of the present invention on adenosine triphosphate and glycogen in gastrocnemius and liver tissues of swimming-bearing mice

As shown in fig. 4 a-D, Adenosine Triphosphate (ATP) and glycogen (glycogen) were significantly increased in both gastrocnemius and liver tissues of mice after the drug treatment of the present invention. The results show that the medicine can effectively regulate the metabolism of protein and amino acid in liver and gastrocnemius tissues, and has better effect than AA.

1.3.5 Effect of the inventive Agents on lactic acid, Urea Nitrogen, Triglycerides and blood glucose in blood of swimming-weight mice

As shown in A-D in FIG. 5, the drug of the present invention can significantly reduce the content of lactic acid, urea nitrogen and triglyceride in blood of mice and increase the blood glucose concentration. The results suggest that the drug of the invention can effectively regulate the metabolism of protein and amino acid in blood, and has better effect than AA.

1.3.6 Effect of the drugs of the present invention on expression of Nrf2 and HO-1 proteins in gastrocnemius and liver tissues of swimming-bearing mice

As shown in fig. 6, the expression of nuclear Nrf2 and HO-1 protein was significantly increased in gastrocnemius muscle and liver tissues in the drug group of the present invention, compared to the control group. The medicine of the invention can prolong the time of heavy swimming of mice and show the anti-fatigue activity by regulating the Nrf2/HO-1 signal conduction path.

1.4. Discussion of the related Art

Fatigue can lead to reduced exercise capacity and reduced work efficiency, and if the exercise cannot be treated in time, chronic fatigue syndrome can be caused, thus threatening the health of human bodies. Because of the unsatisfactory results of current synthetic drugs, Chinese medicines and natural products have attracted more and more attention. The traditional Chinese medicines of astragalus, adenophora tetraphylla and prepared rehmannia root are traditionally used for tonifying qi and nourishing yin. At present, the research on astragalus, adenophora tetraphylla and prepared rehmannia root is mainly focused on the immunoregulatory activity, but the research on the main traditional applications such as qi tonifying and yin nourishing is very little.

We adopt an in vivo model of mouse weight swimming to simulate a verification model of human exercise load. In our study, it was observed that the time for a weight bearing mouse to swim in water was greatly reduced. Compared with the control group of mice, the drug of the invention can remarkably prolong the swimming time of the loaded mice.

Improving the exercise endurance level of the body, reducing the consumption of energy substances and the accumulation of metabolites in the exercise process, and is a basic research mode for searching anti-fatigue active substances. The enzyme and non-enzyme antioxidant can prolong the exercise performance, relieve the fatigue of the body and enhance the activity of antioxidant enzyme, so the body damage caused by over oxidation in the exercise process can be protected, and researches show that the oral antioxidant can prevent or reduce oxidative stress and improve the exercise performance.

Oxidative stress is a key factor in the onset of fatigue. Oxidative stress can damage cellular macromolecules such as DNA, lipids and proteins, which can exacerbate free radical chain reactions, disrupt the integrity of the intestinal mucosal barrier, and activate inflammatory mediators. MDA is thought to be a byproduct of lipid peroxidation, and increased MDA levels cause cross-linking and cytotoxicity of protein and nucleic acid molecules. SOD and GSH-Px are the major antioxidant enzymes in the organism. SOD is a key enzyme, which is converted into H₂O₂(a more stable metabolite) to inactivate superoxide ion, and H₂O₂Further converted to water by catalase or GSH-Px. GSH-Px is an antioxidant enzyme that contributes to the scavenging and inactivation of free radicals, thereby protecting the body from oxidative stress. MDA is an essential cofactor for GSH-Px and SOD and plays a significant role in antioxidation by binding to the active site of GSH-Px. Thus, the measurement of enzyme activity can be interpreted as a performance for assessing fatigue. Our studies indicate that the anti-fatigue effect of the drug of the invention may be closely related to the enhancement of antioxidant enzymes, including CAT, SOD and GSH-Px, and the reduction of MDA level. The results of the study clearly show that the drug of the invention can exert its anti-fatigue effect at least partially by modulating the oxidative stress biomarker.

Nrf2 plays a key role in cellular oxidative stress. Under normal physiological conditions, most of Nrf2 is located in cytoplasm in combination with Kelch-like ECH-associated protein 1(Kelch-like ECH-associated protein 1, Keap1), and Nrf2 fails to enter the nucleus to exert transcriptional activity. Under the condition of oxidative stress, Nrf2 and Keapl ARE dissociated and enter a cell nucleus, and then ARE combined with an Antioxidant Response Element (ARE), so that the transcription and expression of various target genes such as downstream antioxidant proteins, anti-inflammatory factors, II-phase detoxification enzymes and the like ARE started. Activation of an Nrf 2-mediated antioxidant pathway plays an important role in resisting cytotoxic injury, the antioxidant stress effect of an Nrf2 knockout mouse is obviously weakened, and the ROS level is increased. HO-1 is one of downstream target genes regulated by Nrf2, is one of II-phase detoxification enzymes, is also called heat shock protein 32, and plays a key role in resisting oxidative stress injury of cells. In the research, the medicine of the invention can increase the oxidation resistance of liver tissues and gastrocnemius muscles of weight bearing swimming mice by activating Nrf2/HO-1 channel, and regulate the metabolism of amino acid and protein in vivo. The results suggest that the regulatory effect on the Nrf2/HO-1 pathway signaling pathway may be the mechanism of action of the drug of the invention against fatigue.

In conclusion, the medicine provided by the invention has the advantages that the adaptability of the body to exercise load is obviously enhanced, and the possible action mechanism is related to the regulation of Nrf2/HO-1 signal path, the reduction of oxidative stress and the regulation of the metabolism of amino acid and protein in the body.

The foregoing is a more detailed description of the invention in connection with specific/preferred embodiments and is not intended to limit the practice of the invention to those descriptions. It will be apparent to those skilled in the art that various substitutions and modifications can be made to the described embodiments without departing from the spirit of the invention, and these substitutions and modifications should be considered to fall within the scope of the invention.

Claims

1. An anti-fatigue pharmaceutical composition comprises effective components and medically acceptable excipient, and is characterized in that the effective components comprise the following raw materials in percentage by weight:

2. The anti-fatigue pharmaceutical composition according to claim 1, wherein the effective ingredients comprise the following raw materials in percentage by weight: 6 parts of astragalus membranaceus, 6 parts of adenophora tetraphylla and 6 parts of prepared rehmannia root.

3. The anti-fatigue pharmaceutical composition according to claim 1 or 2, wherein the drug is granules, capsules, concentrated pills or tablets.

4. Use of a pharmaceutical composition according to any one of claims 1 to 3 for the preparation of a medicament having an anti-fatigue effect.

5. The preparation method of the anti-fatigue pharmaceutical composition uses effective components and medically acceptable excipient to prepare the pharmaceutical composition, and is characterized in that the effective components comprise the following raw materials in percentage by weight:

6. A process for preparing a pharmaceutical composition according to claim 5, comprising the steps of:

7. The preparation method of the pharmaceutical composition according to claim 6, wherein the ethanol with the mass percentage concentration of 60% is added in the step (1), and the mixture is soaked for 24 hours at normal temperature and ultrasonically extracted for 1 hour.

8. The process for preparing a pharmaceutical composition according to claim 6 or 7, wherein one or more of lactose, dextrin, starch, magnesium stearate, a binder, a solubilizer, a flavoring agent, a bacteriostatic agent, an antioxidant, and a coloring agent are added in step (3).