CN114099616B

CN114099616B - Traditional Chinese medicine composition for resisting long-endurance fatigue

Info

Publication number: CN114099616B
Application number: CN202111168882.9A
Authority: CN
Inventors: 李峰; 何颖; 马捷; 李�杰; 徐一菲; 何青鋆
Original assignee: Beijing University of Chinese Medicine; Chinese Peoples Liberation Army Naval Characteristic Medical Center
Current assignee: Beijing University of Chinese Medicine; Chinese Peoples Liberation Army Naval Characteristic Medical Center
Priority date: 2021-10-08
Filing date: 2021-10-08
Publication date: 2022-12-09
Anticipated expiration: 2041-10-08
Also published as: CN114099616A

Abstract

The invention provides a traditional Chinese medicine composition for resisting fatigue, in particular to long-endurance fatigue, which is prepared from the following raw material medicines: 8-28 parts of astragalus membranaceus, 8-28 parts of fructus aurantii, 5-22 parts of hawthorn, 4-18 parts of dendrobium officinale, 3-15 parts of schisandra chinensis and 2-12 parts of angelica sinensis. The invention also provides a preparation method of the pharmaceutical composition, and the traditional Chinese medicine composition can be used for symptoms such as mental fatigue, hypodynamia, fatigue and the like, and has better effect on common fatigue of long-term aviation personnel. The medicine composition takes soothing the liver and strengthening the spleen as the main treatment principle.

Description

Traditional Chinese medicine composition for resisting long-endurance fatigue

Technical Field

The invention relates to a traditional Chinese medicine composition, in particular to an anti-fatigue traditional Chinese medicine composition, and belongs to the field of medicines.

Background

Long-term fatigue is a physiological psychological phenomenon in which the operating efficiency is obviously reduced due to long-term single or excessive work, nervous psychology, special environment and the like during long-term navigation of a water area, and is mostly manifested as lassitude, inattention, slow response, amnesia, low mood, reduced efficiency and the like. Research shows that the incidence rate of long-endurance fatigue reaches 85.6 percent, which is the main reason for the reduction of the working capacity and the health condition of long-endurance personnel. Therefore, the development of safe and effective anti-fatigue drugs has great significance for guaranteeing the health of long-term aviation personnel. Most of anti-fatigue chemical drugs can generate side effects such as central inhibition and the like, and the advantages of traditional Chinese medicines, especially traditional Chinese medicine and food dual-purpose medicines, are combined, so that the side effects of the medicines can be reduced to the minimum while the medicinal effects are ensured to be exerted, and the safe and effective traditional Chinese medicines are developed to have important significance in relieving long-term fatigue.

There are many hypotheses about the occurrence of fatigue, in which excitatory neurotransmitters in the brain are involved in the maintenance of working ability and efficiency, and in which a reward effect mechanism, which is dominant as the most important excitatory neurotransmitter DA, is involved in the driving of brain work, and when the brain works excessively, its activity is decreased to cause the decrease of motivation, thereby causing the manifestation of fatigue such as the decrease of cognitive function, attention, and strain capacity.

In the nineties of the last century, researchers proved that the content and activity of DA in the brain are closely related to fatigue, and the content of DA in the brain is obviously reduced after exhaustive exercises when the synthesis and metabolism of DA are in a balanced state and the fatigue is delayed. When the body is in a fatigue state and the brain is in a state of relative ischemia and hypoxia, the DRD1 inhibits the level of excitatory neurotransmitter to be in a proper concentration level by regulating a direct path; the DRD2 stimulates the vertebral body system to realize the regulation of the motion level by regulating the indirect path, thereby protecting the body from being damaged.

Therefore, the development of a medicine and food dual-purpose medicine for relieving long-term fatigue symptoms aiming at DA regulation pathway becomes a problem to be solved urgently by technical personnel in the field of fatigue treatment.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide an anti-fatigue traditional Chinese medicine composition and application thereof.

As one aspect of the invention, the invention provides an anti-fatigue traditional Chinese medicine composition, which is prepared from the following raw material medicines: astragalus root, bitter orange, haw, dendrobium officinale, schisandra fruit and Chinese angelica.

Preferably, the traditional Chinese medicine composition is prepared from the following raw material medicines: 8-28 parts of astragalus membranaceus, 8-28 parts of fructus aurantii, 5-22 parts of hawthorn, 4-18 parts of dendrobium officinale, 3-15 parts of schisandra chinensis and 2-12 parts of angelica sinensis;

further preferably, the traditional Chinese medicine composition is prepared from the following raw material medicines: 10-22 parts of astragalus membranaceus, 10-22 parts of fructus aurantii, 6-15 parts of hawthorn, 5-12 parts of dendrobium officinale, 4-10 parts of schisandra chinensis and 3-8 parts of angelica sinensis;

or, the traditional Chinese medicine composition is prepared from the following raw material medicines: 12-18 parts of astragalus membranaceus, 12-18 parts of fructus aurantii, 8-12 parts of hawthorn, 6-10 parts of dendrobium officinale, 5-9 parts of schisandra chinensis and 4-6 parts of angelica sinensis;

most preferably, the traditional Chinese medicine composition is prepared from the following raw material medicines: 15 parts of astragalus membranaceus, 15 parts of fructus aurantii, 10 parts of hawthorn, 8 parts of dendrobium officinale, 7 parts of schisandra chinensis and 5 parts of angelica sinensis;

or 13 parts of astragalus membranaceus, 17 parts of fructus aurantii, 9 parts of hawthorn, 9 parts of dendrobium officinale, 6 parts of schisandra chinensis and 6 parts of angelica sinensis;

or 17 parts of astragalus membranaceus, 13 parts of fructus aurantii, 11 parts of hawthorn, 7 parts of dendrobium officinale, 8 parts of schisandra chinensis and 4 parts of angelica sinensis.

The traditional Chinese medicine composition can be a composition formed by crushing and mixing all the raw material medicines, can also be an extract obtained by mixing or independently extracting all the raw material medicines, or an effective part obtained by further refining and purifying the extract, and can also be a conventional preparation form prepared by adding pharmaceutically acceptable auxiliary materials into the extract/effective part.

Wherein the extraction method comprises decoction extraction, reflux extraction, immersion extraction, ultrasonic extraction, percolation extraction, microwave extraction and the like; the purification method comprises water extraction and alcohol precipitation, alkali dissolution and acid precipitation and various column chromatography purification methods, such as a macroporous resin column, a silica gel column, a reverse phase column and the like; the conventional dosage forms include but are not limited to injections, capsules, tablets, granules, gels, sustained release agents, oral liquids, dropping pills or nano preparations; the pharmaceutically acceptable auxiliary materials comprise: bulking agent, disintegrating agent, lubricant, suspending agent, binder, sweetener, correctant, antiseptic, matrix, etc. The filler comprises: starch, pregelatinized starch, lactose, mannitol, chitin, microcrystalline cellulose, sucrose, etc.; the disintegrating agent comprises: starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, cross-linked sodium carboxymethylcellulose, etc.; the lubricant comprises: magnesium stearate, sodium lauryl sulfate, talc, silica, and the like; the suspending agent comprises: polyvinylpyrrolidone, microcrystalline cellulose, sucrose, agar, hydroxypropyl methylcellulose, and the like; the binder includes starch slurry, polyvinylpyrrolidone, hydroxypropyl methylcellulose, etc.

As another aspect of the present invention, the traditional Chinese medicine composition of the present invention can also use the extract of the raw material medicine as a raw material, so the present invention further provides an anti-fatigue traditional Chinese medicine composition, which is prepared from the following raw materials:

8-28 parts of astragalus extract, 8-28 parts of fructus aurantii extract, 5-22 parts of hawthorn extract, 4-18 parts of dendrobium officinale extract, 3-15 parts of schisandra extract and 2-12 parts of angelica extract;

preferably, the traditional Chinese medicine composition is prepared from the following raw materials:

10-22 parts of astragalus extract, 10-22 parts of fructus aurantii extract, 6-15 parts of hawthorn extract, 5-12 parts of dendrobium officinale extract, 4-10 parts of schisandra extract and 3-8 parts of angelica extract;

further preferably, the traditional Chinese medicine composition is prepared from the following raw materials:

12-18 parts of astragalus extract, 12-18 parts of fructus aurantii extract, 8-12 parts of hawthorn extract, 6-10 parts of dendrobium officinale extract, 5-9 parts of schisandra extract and 4-6 parts of angelica extract;

most preferably, the traditional Chinese medicine composition is prepared from the following raw materials: 15 parts of astragalus extract, 15 parts of fructus aurantii extract, 10 parts of hawthorn extract, 8 parts of dendrobium officinale extract, 7 parts of schisandra extract and 5 parts of angelica extract.

The extract can be water extract or organic solvent extract of each raw material medicine, or refined extract obtained by further refining and purifying the water extract/organic solvent extract.

The organic solvent is selected from one or more of methanol, 20-95% ethanol solution and acetone;

the extraction method for preparing the extract comprises any one of reflux extraction, immersion extraction, ultrasonic extraction or percolation extraction, or the combination of different extraction methods.

The raw material medicaments of the invention, namely astragalus root, bitter orange, hawthorn, dendrobium officinale, chinese magnoliavine fruit and Chinese angelica, all conform to the record of Chinese pharmacopoeia (2020 edition). Wherein, the hawthorn is preferably raw hawthorn.

As another aspect of the invention, the invention also provides application of the traditional Chinese medicine composition in preparing a medicine for resisting long-term fatigue.

The invention has the technical effects that:

in the formula of the invention, the astragalus membranaceus is sweet in taste and slightly warm in nature, enters lung and spleen channels, has the effects of benefiting qi, strengthening exterior, promoting urination, detoxifying and the like, is mainly used for relieving symptoms such as qi deficiency, hypodynamia and the like, has the function of improving body immunity, and is a monarch drug in the formula. The bitter orange is slightly cold in nature and bitter, pungent and sour in taste, and has the effects of regulating qi, relieving chest stuffiness, activating stagnancy and relieving flatulence; the dendrobium nobile is sweet in taste, slightly cold, capable of strengthening yin and replenishing vital essence, capable of thickening intestines and stomach, and deficient in nourishing the interior, and the two medicines are combined to be used as ministerial medicines, and can play an anti-fatigue role together with the astragalus membranaceus and play a role in soothing the liver and regulating the flow of qi. The angelica has sweet and warm taste, and has the functions of relaxing bowel, promoting blood circulation and removing blood stasis, and the angelica enters heart, liver and lung channels; the hawthorn is sweet and sour in taste, neutral in nature, and capable of entering spleen channels, strengthening spleen, promoting digestion, dissipating stagnation of qi and promoting blood circulation; the schisandra chinensis is sour in taste and warm in nature, and has the effects of soothing liver, tonifying spleen, regulating qi, strengthening body resistance and relieving fatigue by combining various medicines.

The formula of the invention is further optimized by the inventor on the basis of earlier research results, and compared with the earlier formula, the traditional Chinese medicine composition of the invention has the advantages of ensuring the clinical efficacy and improving the safety. In an acute toxicity experiment, 113.5 times of dose of the traditional Chinese medicine composition and a control medicine are given to adults daily, the average value of the contents of glutamic-pyruvic transaminase, glutamic-oxaloacetic transaminase and urea nitrogen of a control group mouse is higher than that of the traditional Chinese medicine composition, the control group also has the condition that one mouse dies, and the indexes of the dead mouse glutamic-pyruvic transaminase, the glutamic-oxaloacetic transaminase and the urea nitrogen are obviously improved compared with those of a normal group mouse. The above results indicate that the side effects of the control drug on liver and kidney functions are greater than those of the traditional Chinese medicine composition of the invention. Therefore, from the perspective of medication safety, the traditional Chinese medicine composition is more suitable for long-term administration, has low toxic and side effects, and is safe and effective.

Detailed Description

Example 1

The formula is as follows: 15g of astragalus membranaceus, 15g of fructus aurantii, 10g of hawthorn, 8g of dendrobium officinale, 7g of schisandra chinensis and 5g of angelica sinensis;

the preparation method comprises the following steps: taking the raw materials in proportion, decocting in water twice for 40min and 30min, filtering, collecting water decoction, and concentrating to obtain extract;

and preparing the extract into tablets according to the conventional tablet preparation process.

Example 2

The formula is as follows: 13g of astragalus membranaceus, 17g of fructus aurantii, 9g of hawthorn, 9g of dendrobium officinale, 6g of schisandra chinensis and 6g of angelica sinensis;

the preparation method comprises the following steps: taking the raw materials in proportion, adding 50% ethanol, reflux extracting twice, the first time for 40min. Filtering for 20min, mixing extractive solutions, and concentrating to obtain extract;

and preparing the extract into granules according to the conventional granule preparation process.

Example 3

The formula is as follows: 17g of astragalus membranaceus, 13g of fructus aurantii, 11g of hawthorn, 7g of dendrobium officinale, 8g of schisandra chinensis and 4g of angelica sinensis;

the preparation method comprises the following steps: the raw materials are taken according to the proportion, and are decocted and extracted twice with water for 40min for the first time. Filtering for 20min, mixing decoctions, and concentrating to obtain extract;

and preparing the extract into capsules according to the conventional capsule preparation process.

Example 4

The formula is as follows: 11g of astragalus membranaceus, 20g of fructus aurantii, 7g of hawthorn, 11g of dendrobium officinale, 5g of schisandra chinensis and 8g of angelica sinensis;

the preparation method comprises the following steps: taking the raw materials in proportion, adding 60% ethanol, reflux extracting twice, the first time for 40min. Filtering for 20min, mixing extractive solutions, and concentrating to obtain extract;

and preparing the extract into honeyed pills according to a conventional honeyed pill preparation process.

Example 5

The formula is as follows: 21g of astragalus membranaceus, 11g of fructus aurantii, 14g of hawthorn, 6g of dendrobium officinale, 9g of schisandra chinensis and 4g of angelica sinensis;

the extract is prepared into powder according to the conventional powder preparation process.

Example 6

The formula is as follows: 9g of astragalus membranaceus, 25g of fructus aurantii, 6g of hawthorn, 16g of dendrobium officinale, 4g of schisandra chinensis and 10g of angelica sinensis;

the preparation method comprises the following steps: taking the raw materials in proportion, adding 60% ethanol, and ultrasonically extracting twice for 40min for the first time. Filtering for 20min, mixing extractive solutions, and concentrating to obtain extract;

the extract is prepared into dripping pills according to the conventional dripping pill preparation process.

Example 7

The formula is as follows: 26g of astragalus membranaceus, 9g of fructus aurantii, 20g of hawthorn, 5g of dendrobium officinale, 14g of schisandra chinensis and 3g of angelica sinensis;

the preparation method comprises the following steps: the raw materials are taken according to the proportion, and water is added for reflux extraction twice, and the first time is 40min. Filtering for 20min, mixing extractive solutions, and making into oral liquid by conventional oral liquid preparation method.

Example 8

The formula is as follows: 15g of astragalus extract, 15g of fructus aurantii extract, 10g of hawthorn extract, 8g of dendrobium officinale extract, 7g of schisandra extract and 5g of angelica extract.

Example 9

The formula is as follows: 13g of astragalus extract, 17g of fructus aurantii extract, 9g of hawthorn extract, 9g of dendrobium officinale extract, 6g of schisandra extract and 6g of angelica extract.

Example 10

The formula is as follows: 17g of astragalus extract, 13g of fructus aurantii extract, 11g of hawthorn extract, 7g of dendrobium officinale extract, 8g of schisandra extract and 4g of angelica extract.

Example 11

The formula is as follows: 11g of astragalus extract, 20g of fructus aurantii extract, 7g of hawthorn extract, 11g of dendrobium officinale extract, 5g of schisandra extract and 8g of angelica extract.

Example 12

The formula is as follows: 21g of astragalus extract, 11g of fructus aurantii extract, 14g of hawthorn extract, 6g of dendrobium officinale extract, 9g of schisandra extract and 4g of angelica extract.

Example 13

The formula is as follows: 9g of astragalus extract, 25g of fructus aurantii extract, 6g of hawthorn extract, 16g of dendrobium officinale extract, 4g of schisandra extract and 10g of angelica extract.

Example 14

The formula is as follows: 26g of astragalus extract, 9g of fructus aurantii extract, 20g of hawthorn extract, 5g of dendrobium officinale extract, 14g of schisandra extract and 3g of angelica extract.

The extracts described in examples 8-14 above are water extracts of the respective crude drugs. The extracts are combined according to the formula amount and then prepared into conventional oral dosage forms such as tablets, capsules, powder, granules, honeyed pills, dripping pills, oral liquid and the like according to the conventional preparation process.

Example 15 acute toxicity test

1 test materials

1.1 animals and groups

40 healthy 4-week-old Kunming mice with male sex, SPF grade and body weight (20 +/-2) g are selected and ordered from Sibefu (Beijing) laboratory animal science and technology Limited, and the license number of the laboratory animal is (SCXK (Beijing) 2016-0002). Animals were kept in an animal house at a temperature of 23 + -1 deg.C and a relative humidity of 50%. After 5 days of adaptive feeding, the feed is randomly divided into a normal group, an experimental group, a control group and a western medicine group by using a random number table, wherein each group comprises 10 animals.

1.2 Experimental drugs

Experimental drugs:

the preparation method comprises the following steps: taking the raw materials according to a certain proportion, adding water, decocting twice, the first time for 40min, the second time for 30min, filtering, taking water decoction, concentrating to obtain extract;

control drugs: 15g of immature bitter orange, 15g of astragalus, 5g of angelica, 10g of hawthorn, 10g of radix bupleuri and 5g of gardenia; the extract is prepared by the same method as the experimental medicine.

Positive control drug: coenzyme Q10 (product company: manufactured by Weishu (China) pharmaceutical industry Co., ltd., approved article number: national Standard H10930021) provided by pharmacy of Orthon Hospital, the first clinical college of medicine university, beijing.

The experimental drug is concentrated to the maximum concentration (equivalent to 2g/ml of crude drug, calculated when the liquid medicine can be just pressed out by a gavage needle), the preparation method of the contrast drug is the same as that of the experimental drug, each group of mice is fasted for 12 hours without water prohibition before administration, then the maximum administration method is adopted, the experimental drug of 4ml/100g of the experimental group and the contrast drug of 4ml/100g of the contrast group are administered in the maximum administration volume within 24 hours, and the gavage is carried out for 3 times. The normal group was given the same volume of distilled water and the gavage was continued for 20 days. The western medicine group is administered with coenzyme Q10 of 1ml/100g by intragastric administration.

1.3 Experimental instrumentation

Frying pan Oubei

Crystal macro BCD-256WDGH of common refrigerator

Electronic balance German Sadolis BSA224S

2 index of experiment

2.1 Observation of toxic reactions in mice

2.2 record the physical and physical conditions of the mice

2.3 detecting the contents of glutamic-pyruvic transaminase, glutamic-oxalacetic transaminase and urea nitrogen in each group of mice.

3. Results of the experiment

3.1 mouse toxicity response assessment

The experimental and control groups use 113.5 times of the daily dose of experimental and control drugs for adults for intragastric administration, and the normal group uses the same volume of physiological saline. No adverse reactions such as listlessness, toxic symptoms, death and the like appear in mice in experimental groups from the time of administration to the end of experiments. In the evaluation of each important system, no phenomena such as obvious sleepiness, dysphoria, irritability, coma and the like are seen in the aspect of the nervous system, no obvious stereotypy movement, forced movement, muscle relaxation and paralysis are seen, and no obvious enlargement and reduction of the pupil are seen; no obvious cough is seen in the aspect of respiratory system, and no abnormal secretion is seen in the mouth and nose; in the aspect of the cardiovascular system, no obvious varicose veins are seen on the whole body, and no obvious symptoms of accelerated or slowed heart rate and no obvious bleeding are seen in precordial palpation; vomiting, abdominal distension, abdominal tension and abnormal contraction are not seen in the aspect of digestive system, loose stool is not seen obviously, and redness and ulcer are not seen around the anus; no obvious perineum, labial and breast swelling is seen in the urogenital system; the skin of the whole body does not have congestion, cyanosis, pale, rash and loose hair; abnormal lacrimation, eyelid ptosis, eyeball protrusion and tremor are not seen in the eyes; no ulcer and defect were observed on the ear and nose. 1 case of death of the control group mice occurs, and indexes of the dead mice, such as glutamic-pyruvic transaminase, glutamic-oxalacetic transaminase, urea nitrogen and the like, which reflect the functions of the liver and the kidney are obviously improved compared with the normal group mice. No significant abnormalities were seen in the remaining mice and no significant abnormalities were observed in the evaluation of each system.

3.2 mouse body Mass variation

The results are shown in Table 1.

No statistical difference was observed between the initial body weight and the final body weight of each group of mice (P > 0.05)

Table 1 initial and final body weight tables of mice (n,

)

group of	Initial body Mass (g)	Last body mass (g)
			Normal group	19.40±0.70	36.70±3.56
Control group	19.50±0.71	37.70±2.83
			Experimental group	19.10±0.57	37.60±1.90
Western medicine group	19.80±1.32	38.0±1.83

3.3 serum glutamic-pyruvic transaminase, glutamic-oxalacetic transaminase and urea nitrogen content of mice

The results are shown in Table 2.

The contents of glutamic-pyruvic transaminase, glutamic-oxalacetic transaminase and urea nitrogen in mice in each group are compared pairwise, the difference among the groups has no statistical significance (P is more than 0.05), but the average values of the contents of the glutamic-pyruvic transaminase, the glutamic-oxalacetic transaminase and the urea nitrogen in the control group of mice are higher than those of the experimental group and the normal group of mice.

Table 2 serum glutamic-pyruvic transaminase, glutamic-oxalacetic transaminase, urea nitrogen content comparison of mice (n,

)

from the acute toxicity test results, the content average values of glutamic-pyruvic transaminase, glutamic-oxalacetic transaminase and urea nitrogen of the mice in the control group are higher than those of the mice in the experimental group under the same dosage, and the control group also has the condition that one mouse dies, so that the indexes of the glutamic-pyruvic transaminase, the glutamic-oxalacetic transaminase and the urea nitrogen of the dead mice are obviously improved compared with those of the mice in the normal group. Glutamic-pyruvic transaminase, glutamic-oxalacetic transaminase and urea nitrogen are indexes reflecting liver and kidney functions. The results show that the control medicament has more side effects on the liver and kidney functions than the experimental medicament, so the experimental medicament is more suitable for long-term administration from the perspective of medication safety, has low toxic and side effects, and is safe and effective.

EXAMPLE 16 drug efficacy test

1 test materials

1.1 animals and groups

Healthy adult male SD rats 60 with body weight 210 + -10 g, SPF rating, purchased 3 days in advance and bred adaptively. The experimental animals were purchased from sbefu (beijing) experimental animal science and technology ltd; the license number is SCXK (Jing) 2016-0002. Feeding in animal room with temperature (23 + -1) ° C and relative humidity of 50%. The method is divided into 4 groups according to a random digital table method, wherein the 4 groups are respectively a normal group, a long-endurance fatigue model group, an experimental group and a western medicine group, and each group comprises 15 medicines.

1.2 Experimental drugs

(1) Experimental drug 1: example 1 a drug extract was prepared, and the above crude drugs were all from beijing Tongrentang pharmaceutical industry co.

(2) Western medicines: coenzyme Q10 (product company: manufactured by Weishu (China) pharmaceutical industry Co., ltd., approved article number: national Standard H10930021) provided by pharmacy of Orthon Hospital, the first clinical college of medicine university, beijing.

1.3 Experimental instrumentation

2 method of experiment

2.1 model replication

Adaptive feeding: the animals are purchased and are adaptively raised for 3 days, and the adaptive raising is carried out in a traditional Chinese medicine diagnostic animal research laboratory. 40W daylight lamp irradiation (8. Feeding with conventional murine feed.

The molding method comprises the following steps: the rat is placed in a water surface shaking platform instrument every day to stand for 2 hours, a wave maker is set to be 85 r/min, the rat is placed in a water environment small platform sleep deprivation box (MMPM) from 8 pm to 6 pm in the morning for 10 hours in total, a mold is made for 12 hours every day, a water bottle and food are placed in a gap above the model during the mold making period, the rat can drink water normally after eating, and the mold is made continuously for 21 days.

2.2 dosing regimens

And (3) beginning to administer corresponding traditional Chinese medicines and western medicines to the rats on the 15 th day of molding, converting the dosage of the human body into the equivalent dosage of the rats as the dosage of the rats, and controlling the intragastric volume to be 1mL/100g. The rest groups were drenched with equal volume of distilled water.

2.3 detection criteria and methods

(1) Taking materials

After the molding is finished, performing abdominal anesthesia by using 10% chloral hydrate at the concentration of 0.3ml/100g, cutting the head, placing on ice, taking out the striatum, rapidly transferring to liquid nitrogen for freezing preservation, and storing in a refrigerator at the temperature of minus 80 ℃ for later use when the surface color of the brain tissue becomes white.

(2) Experimental procedure

I Gene level detection

1.1 extraction of Total RNA from samples

Taking the striatum obtained by material drawing out from an ultra-low temperature refrigerator at minus 80 ℃, and carrying out experimental operation according to the instruction of the Hipure Total RNA Mini Kit, wherein the specific process is as follows:

(1) pretreatment: adding 4 times of volume of absolute ethyl alcohol into Buffer RW 2; preparing 70% ethanol by using absolute ethanol and DEPC (diethyl ether-diethyl ether) treated water; a small amount of antifoam was added to Buffer RL.

(2) Buffer RL 750ul was added to each tissue sample.

(3) Tissue samples were disrupted using an ultrasonicator on ice at 10000r/min and centrifuged for 5min.

(4) The gDNA Filter Mini Column was placed in a 2ml collection tube and the supernatant from the previous step was transferred to the Column.

(5) 10000r/min for 2min, discarding the gDNA Filter Mini Column.

(6) Adding 750ul of 70% ethanol prepared from DEPC treated water into the obtained filtrate, and blowing and beating for 5 times to obtain a mixed solution.

(7) Hipure RNA Mini Column was placed in a 2ml collection tube.

(8) Adding 750ul of the mixed solution obtained in the previous step, centrifuging at 10000r/min for 1min, and discarding the waste liquid obtained by centrifuging.

(9) Adding the rest 750ul of the mixed solution obtained in the previous step again at 10000r/min, centrifuging for 1min, and discarding the waste liquid obtained by centrifugation.

Adding 500ul Buffer RW2 in the R, centrifuging for 1min at 10000r/min, and discarding the waste liquid obtained by centrifugation.

Adding 500ul Buffer RW1, centrifuging at 10000r/min for 1min, and discarding the waste liquid obtained by centrifugation.

The empty column was centrifuged for 2min and the HipureRNAMiniColumn column was transferred to a 1.5ml centrifuge tube.

50ul RNase FreeWater was added and left to stand for 2min.

Centrifuging at 10000r/min for 1min to obtain total RNA.

After RNA is obtained, the concentration of the sample is detected by using an ultraviolet spectrophotometer, and the values of A260/280 and A260/230 are read.

1.2 Reverse transcription of RNA

(1) According to the concentration of the extracted RNA, the volume of 2ug of RNA, the amount of DEPC treatment water required for preparing 20ul of reaction system, and the addition amounts of template RNA and DEPC treatment water are calculated.

A reverse transcription reaction system is prepared by using a THERMO reverse transcription kit, the addition amount of each reagent is shown in table 3, and after all samples and reagents are added, eight rows are centrifuged for a short time.

TABLE 3 20ul reverse transcription System

Name(s)	Adding amount of
		RT	1ul
RiboLockTM RNase inhibitor	1ul
		5×Reaction Buffer	4ul
dNTP	2ul
		Oligo(dT)18	1ul
Random Hexamer Primer	1ul
		RNA solution	2ug
DEPC treated water	Make up the total reaction system to 20ul

The reverse transcription program is set as follows: keeping the temperature at 42 ℃ for 60min, keeping the temperature at 70 ℃ for 5min, cooling to-12 ℃, and storing the obtained cDNA in a refrigerator at-80 ℃.

1.3 real-time fluorescent quantitative PCR amplification

(1) According to the design of the experiment, shanghai bioengineering, inc. was entrusted with the synthesis and verification of primers, the sequences of which are shown in the following Table, and the sequences of the primers used in the experiment are shown in Table 4.

TABLE 4 primer sequences

The cDNA, primers, SYBR Green Realtime PCR Master Mix, DEPC treated water were prepared in 20ul reaction system (Table 5) with 3 wells per well.

TABLE 5 RT-PCR 20ul reaction System

Name (R)	Addition amount (ul)
		cDNA	3
SYBR Green Realtime PCR Master Mix	10
		Forward direction of primer	0.5
Primer reverse direction	0.5
		DEPC water	6

(3) Use of

Real-time PCR instrument for amplification, the program set up is shown in Table 6; obtaining the CT value of each hole after the completion, if no obvious difference exists among the auxiliary holes, taking the average value of 3 auxiliary holes as the CT value of the sample, and adopting 2- ^△△ And calculating the ratio of the target gene to the reference gene beta-actin by a CT method.

TABLE 6 PCR amplification Programming

1.4 data processing

Data were statistically processed using SPSS17.0 software. In each index, the measurement variable adopts the mean value plus or minus standard deviation

Describing, normally distributed data adopts single-factor variance analysis, and LSD-t test is carried out by pairwise comparison. And comparing the data distributed in a skewed state among groups by adopting a rank sum test. The inspection level is taken as alpha =0.05<0.05 is a significant difference, P<0.01 is very significant difference and has statistical significance.

II protein level detection

Mixing Ripa (Radio immunopotentiation Assay) lysate and a protease inhibitor phenylmethylsulfonyl fluoride (PMSF) uniformly according to a ratio of 1000.

2.1 Total protein concentration determination

The BCA (bicinchoninic acid) method is used for detecting the total protein concentration, and the determination process mainly comprises the following steps:

(1) preparing a working solution: the BCA working solution was prepared by adding 50 volumes of the BCA reagent to 1 volume of the Cu2+ reagent (50.

(2) Dilution of the standard: mu.L of BSA standard was diluted to 100. Mu.L with PBS to a final concentration of 0.5mg/mL. The standard was added to the protein standard wells of a 96-well plate at 0,2,4,6,8,12,16,20. Mu.L, and PBS was made up to 20. Mu.L.

(3) Dilution of the sample: representative samples were diluted 2,4, 8, 16 and stock and dilution were added to 20. Mu.L to sample wells of a 96-well plate.

(4) 200. Mu.L of the BCA working solution was added to each well, and the mixture was left in a 37 ℃ dry box for 30 minutes. And (3) measuring the OD value of A562nm by using a microplate reader, calculating the protein concentration according to the standard curve, and determining the appropriate dilution factor.

(5) After all samples are diluted correspondingly, the steps are repeated to detect the protein concentration of each sample.

2.2 preparation of electrophoretic gels

According to the results of the preliminary experiment and the molecular weight of the primary antibody of the experiment, 10 percent separation gel is prepared. 4.86ml of deionized water, 3ml of a gel separation buffer solution, 4ml of a 30% acrylamide solution, 10% AP100ul and 8ul of TEMED were added to each beaker, and the mixed solution in the beaker was rapidly and sufficiently mixed. Then, using a 1000ul micropipette to add the solution to the glass plate on the gel making frame until the solution is added to the green wicket of the gel making frame by 2-3mm, then adding deionized water gently to ensure that water flow impacts on the long plate and flows onto the gel slowly until the glass plate is filled to isolate air, and sucking out the solution by using a thin gun head when bubbles appear. Standing at room temperature for 30min. Then preparing 5% concentrated glue, pouring off the deionized water in the glass plate, adding 2.87ml of deionized water, 1.25ml of concentrated glue buffer solution and 0.83ml of 30% acrylamide into a beaker in sequence, cutting filter paper into strips of 1cmX4cm, stretching the strips into the glass plate of a glue making frame, sucking excess water, then putting 10 AP50ul and TEMED5ul into the beaker, quickly mixing the mixture, adding the mixed concentrated glue mixed solution into the glass plate of the glue making frame by using a 1000ul micropipettor, quickly vertically inserting a comb into the glass plate after the mixture is filled, finally supplementing a proper amount of concentrated glue into the glass plate, and standing the glass plate at room temperature for 30min.

2.3 preparation of Loading buffer

Based on the protein concentration measured by BCA method, all sample protein concentrations were adjusted to the same level by adding PBS, according to the protein solution volume: loading buffer =4 ratio of 1 loading buffer was added, sealed and boiled to cool quickly.

2.4 electrophoresis

Filling the inner tank of the electrophoresis device with 1 × electrophoresis buffer solution, and adding the rest electrophoresis solution into the outer tank after ensuring no leakage. The gel comb was pulled out vertically upwards with both hands gently, 4ul Marker was added to both sides, and 6ul sample was added to each remaining well. Electrifying and pressurizing to 80V for electrophoresis until the blue sample buffer solution reaches the bottom, and turning off the power supply.

2.5 transfer film

And (3) placing the prepared 1 Xelectrotransfer buffer solution into a refrigerator for precooling 2h before membrane conversion, taking out the electrotransfer solution half an hour before membrane conversion, and soaking the sponge pad and the filter paper used in the membrane conversion process. Before starting to rotate the membrane, the PVDF membrane is cut to be a proper size and is soaked in methanol for 1min for activation. Opening the clamping plate for film transfer, respectively laying a sponge pad and filter paper, laying a gel block obtained by electrophoresis on a black surface, laying a PVDF film activated by methanol on a transparent surface, and pressing layer by using a glass rod. The black surface of the rotating film clamp is put in corresponding to the black surface of the electrophoresis box, crushed ice is put outside the electrophoresis box, and the film is rotated for 90min under the voltage of 80V.

2.6 sealing

And (3) cleaning the PVDF membrane after membrane conversion for 2 times by using 1xTBST, 5min each time, immersing the PVDF membrane in 5% skimmed milk powder after the PVDF membrane is cleaned, sealing the PVDF membrane, and placing the PVDF membrane in a shaking table for 1h at normal temperature.

2.7 incubation Primary antibody

The blocked PVDF membrane was washed 5 times with 1XTBST for 5min each time and gently shaken on a shaker. Diluting the primary antibody by using an antibody diluent by the following dilution times respectively: DRD1=1, DRD2r1, 500, DRD5= 1. PVDF membrane was immersed in primary anti-diluent at room temperature for 90min and placed on a shaker with gentle shaking.

2.8 incubation Secondary antibodies

After the end of the incubation of the primary antibody, the PVDF membrane was washed 5 times for 5min each and gently shaken on a shaker. The secondary antibody was diluted with 5% nonfat dry milk, and the PVDF membrane was immersed at room temperature for 1 hour and gently shaken on a shaker.

2.9ECL coloration

After incubation with the secondary antibody, the PVDF membrane was washed 5 times for 5min each and gently shaken on a shaker. A color developing solution (solution a: solution B = 1. Spreading a preservative film, placing the PVDF film on the preservative film with the front side upward, flatly spreading and dripping the developing solution on the PVDF film, averaging 200ul of each film, and shaking to remove bubbles to ensure that the PVDF film is completely covered by the developing solution. During pre-testing, gradient exposure is used, and after the optimal exposure time is found, a picture is taken.

2.10 data processing

Describing, normally distributed data adopts single-factor variance analysis, and LSD-t test is carried out by pairwise comparison. The data of the skewed distribution is compared among groups by adopting a rank sum test. The inspection level is taken as alpha =0.05<0.05 is a significant difference, P<0.01 is very significant difference and has statistical significance.

3 results of the experiment

3.1 rat striatum DRD1, DRD2mRNA Gene expression results

The results are shown in Table 7.

TABLE 7 rat striatum DRD1, DRD2mRNA gene expression results

P < 0.05 × P < 0.01 in comparison with normal group, and in comparison with model group ^# ＜0.05

The real-time fluorescent quantitative PCR experiment result shows that the data of rat striatum DRD1 and DRD2 are statistically different, the gene expression of 2-index long-range fatigue model groups is lower than that of a normal group, and the gene expression of an experimental group is higher than that of the model group and is superior to that of a positive control drug.

3.2 expression results of DRD1 and DRD2 proteins in rat striatum

The results are shown in Table 8.

The results of the Westernblot experiment show that the data of rat striatum DRD1 and DRD2 are statistically different, the long-endurance fatigue model histone expression of 2 indexes is lower than that of the normal group, and the experimental histone expression is higher than that of the model group.

TABLE 8 expression results of rat striatum DRD1 and DRD2 proteins

Group of	DRD1	DRD2
			Normal group	1.05±0.1	1.17±0.08
Model set	0.48±0.17**	0.61±0.11**
			Experimental group	0.94±0.15 ^##	1±0.1 ^##
Western medicine group	1.01±0.14 ^##	1.14±0.1 ^##

P < 0.01 in comparison with normal group, and in comparison with model group ^## P＜0.01

When the body is in a fatigue state and the brain is in a state of relative ischemia and hypoxia, the DRD1 inhibits the level of excitatory neurotransmitter to be in a proper concentration level by regulating a direct path; the DRD2 stimulates the vertebral body system to realize the regulation of the motion level by regulating the indirect path, thereby protecting the body from being damaged. The traditional Chinese medicine composition can obviously improve the gene and protein expression of the DRD1 and DRD2 of the long-range model rats, and shows that the traditional Chinese medicine composition can relieve long-range fatigue symptoms aiming at DA regulating channels.

Example 17 clinical observations

1 test drugs: the tablets prepared in example 1 of the present invention, one tablet at a time, twice a day;

2, patient statistics: the experimenter is a long-term maritime navigation person, and the number of the experimenters is 160, wherein 8 severely tired people have symptoms of severe lassitude and hypodynamia, incapability of concentrating attention and the like in long-term navigation; for 34 persons with moderate fatigue, the limbs of the person feel tired in long voyage; in the case of the mild fatigue, 118 people had slight lassitude and lack of strength during long-term flight.

3 results of the treatment

The results are shown in Table 9.

TABLE 9 clinical observation result of the traditional Chinese medicine composition for relieving long-term fatigue

Person(s)	Severe fatigue	Moderate fatigue	Mild fatigue
					8	34	118
Relief of symptoms	5	21	110
				Symptom reduction	3	10	8
Nullification	0	3	0

The results show that the total 136 patients with symptom relief account for 85 percent; the total of 21 patients with reduced symptoms accounts for 13.125 percent; the number of the ineffective people is 3, and the ineffective people account for 1.875 percent. The total effective rate is 98.125%. The traditional Chinese medicine composition can obviously relieve long-term fatigue.

Claims

1. The traditional Chinese medicine composition for resisting long-endurance fatigue is characterized by being prepared from the following raw material medicines: 8-28 parts of astragalus membranaceus, 8-28 parts of fructus aurantii, 5-22 parts of hawthorn, 4-18 parts of dendrobium officinale, 3-15 parts of schisandra chinensis and 2-12 parts of angelica sinensis.

2. The traditional Chinese medicine composition of claim 1, wherein the traditional Chinese medicine composition is prepared from the following raw materials: 10-22 parts of astragalus membranaceus, 10-22 parts of fructus aurantii, 6-15 parts of hawthorn, 5-12 parts of dendrobium officinale, 4-10 parts of schisandra chinensis and 3-8 parts of angelica sinensis.

3. The traditional Chinese medicine composition of claim 2, wherein the traditional Chinese medicine composition is prepared from the following raw material medicines: 12-18 parts of astragalus membranaceus, 12-18 parts of fructus aurantii, 8-12 parts of hawthorn, 6-10 parts of dendrobium officinale, 5-9 parts of schisandra chinensis and 4-6 parts of angelica sinensis.

4. The traditional Chinese medicine composition of claim 3, wherein the traditional Chinese medicine composition is prepared from the following raw materials: 15 parts of astragalus membranaceus, 15 parts of fructus aurantii, 10 parts of hawthorn, 8 parts of dendrobium officinale, 7 parts of schisandra chinensis and 5 parts of angelica sinensis;

5. The traditional Chinese medicine composition of any one of claims 1 to 4, wherein the traditional Chinese medicine composition is a composition prepared by crushing and mixing raw materials, or an extract prepared by mixing or extracting the raw materials separately, or an effective part prepared by further refining and purifying the extract, or a conventional preparation prepared by adding pharmaceutically acceptable auxiliary materials.

6. The traditional Chinese medicine composition for resisting long-term fatigue is characterized by being prepared from the following raw materials: 8-28 parts of astragalus extract, 8-28 parts of fructus aurantii extract, 5-22 parts of hawthorn extract, 4-18 parts of dendrobium officinale extract, 3-15 parts of schisandra extract and 2-12 parts of angelica extract; wherein the extracts are water extracts of the raw material medicines respectively.

7. The traditional Chinese medicine composition of claim 6, wherein the traditional Chinese medicine composition is prepared from the following raw materials: 10-22 parts of astragalus extract, 10-22 parts of fructus aurantii extract, 6-15 parts of hawthorn extract, 5-12 parts of dendrobium officinale extract, 4-10 parts of schisandra extract and 3-8 parts of angelica extract.

8. The use of the Chinese medicinal composition of any one of claims 1-4 and 6-7 in the preparation of a medicament for resisting long-term fatigue.