阅读说明：本技术 一种化合物、中药益生菌发酵产物及其在制备具有抗疲劳作用的药物或保健品中的应用 (Compound, traditional Chinese medicine probiotic fermentation product and application of compound and traditional Chinese medicine probiotic fermentation product in preparation of anti-fatigue medicin) 是由 刘志刚 刘杰 刘晓宇 于 2019-11-26 设计创作，主要内容包括：本发明涉及生物医学技术领域,具体公开了一种化合物、中药益生菌发酵产物及其在制备具有抗疲劳作用的药物或保健品中的应用。所述的中药益生菌发酵产物,包含式(Ⅰ)化合物；其制备方法包含如下步骤：(1)取中药原料用益生菌进行发酵,得发酵液；(2)将发酵液进行干燥后即得所述的中药益生菌发酵产物；步骤(1)中所述的中药原料为包含龙眼肉、党参、黄芪、当归、枸杞子、金银花和白术的中药原料。本发明所述的式(Ⅰ)化合物以及中药益生菌发酵产物具有优异的抗疲劳作用；可以用于制备药物和保健品。(The invention relates to the technical field of biomedicine, and particularly discloses a compound, a traditional Chinese medicine probiotic fermentation product and application of the compound and the traditional Chinese medicine probiotic fermentation product in preparation of a medicine or a health-care product with an anti-fatigue effect. The traditional Chinese medicine probiotic fermentation product comprises a compound shown in a formula (I); the preparation method comprises the following steps: (1) taking the traditional Chinese medicine raw materials, and fermenting with probiotics to obtain fermentation liquor; (2) drying the fermentation liquor to obtain the traditional Chinese medicine probiotic fermentation product; the traditional Chinese medicine raw materials in the step (1) are traditional Chinese medicine raw materials containing arillus longan, radix codonopsis, radix astragali, angelica sinensis, fructus lycii, honeysuckle and bighead atractylodes rhizome. The compound of the formula (I) and the traditional Chinese medicine probiotic fermentation product have excellent anti-fatigue effect; can be used for preparing medicines and health products.)

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof;

2. a process for the preparation of a compound of formula (i) as claimed in claim 1, comprising the steps of:

(1) taking the traditional Chinese medicine raw materials, and fermenting with probiotics to obtain fermentation liquor;

(2) drying the fermentation liquor to obtain a crude product;

(3) separating the crude product by chromatography to obtain a compound of formula (I);

the traditional Chinese medicine raw materials in the step (1) are traditional Chinese medicine raw materials containing arillus longan, radix codonopsis, radix astragali, angelica sinensis, fructus lycii, honeysuckle and bighead atractylodes rhizome;

the probiotics in the step (1) are selected from two or more than two of bifidobacterium infantis, lactobacillus acidophilus, bifidobacterium bifidum and lactobacillus salivarius.

3. The preparation method according to claim 2, wherein the specific method of step (1) is: extracting the Chinese medicinal materials with water, and concentrating the extractive solution to obtain concentrated solution; then adding a fermentation nutrient into the concentrated solution; and then adding probiotics for fermentation for 36-60 h under the conditions that the temperature is 37-42 ℃ and the pH is 6.5-7.5 to obtain fermentation liquor.

4. The process according to claim 2, wherein the chromatographic technique in step (3) is a preparative HPLC technique; the specific conditions of the preparative HPLC technique are as follows: adopting a reversed phase chromatographic column; selecting an ultraviolet detector, wherein the detection wavelength is 220-230 nm; using a trifluoroacetic acid aqueous solution with the volume percent of 0.05-0.2 as a mobile phase A, using a trifluoroacetic acid acetonitrile solution with the volume percent of 0.05-0.2 as a mobile phase B, wherein the mobile phase A: mobile phase B85: 15; and (3) collecting the part corresponding to the chromatographic peak for 8.0-9.0 min, and concentrating and drying to obtain the compound shown in the formula (I).

5. A traditional Chinese medicine probiotic fermentation product is characterized by comprising a compound shown in a formula (I).

6. The method for preparing the traditional Chinese medicine probiotic fermentation product according to claim 5, characterized by comprising the following steps:

(1) taking the traditional Chinese medicine raw materials, and fermenting with probiotics to obtain fermentation liquor;

(2) drying the fermentation liquor to obtain the traditional Chinese medicine probiotic fermentation product;

the traditional Chinese medicine raw materials in the step (1) are traditional Chinese medicine raw materials containing arillus longan, radix codonopsis, radix astragali, angelica sinensis, fructus lycii, honeysuckle and bighead atractylodes rhizome;

the probiotics in the step (1) are selected from two or more than two of bifidobacterium infantis, lactobacillus acidophilus, bifidobacterium bifidum and lactobacillus salivarius.

7. The preparation method according to claim 5, wherein the specific method of step (1) is: extracting the Chinese medicinal materials with water, and concentrating the extractive solution to obtain concentrated solution; then adding a fermentation nutrient into the concentrated solution; and then adding probiotics for fermentation for 36-60 h under the conditions that the temperature is 37-42 ℃ and the pH is 6.5-7.5 to obtain fermentation liquor.

8. Use of a compound of formula (i) as defined in claim 1 or a pharmaceutically acceptable salt thereof or a fermentation product of a probiotic of a traditional Chinese medicine as defined in claim 6 in the manufacture of a medicament or a health product.

9. The use according to claim 8, wherein the medicament is a medicament having an anti-fatigue effect; the health care product has the function of resisting fatigue.

10. The use according to claim 9, wherein the medicament or health product comprises a compound of formula (i) or a pharmaceutically acceptable salt thereof or a probiotic fermentation product of a traditional Chinese medicine and a carrier; the mass fraction of the compound of formula (I) or the medicinal salt thereof or the traditional Chinese medicine probiotic fermentation product is 5-95%.

Technical Field

The invention relates to the technical field of biomedicine, in particular to a compound, a traditional Chinese medicine probiotic fermentation product and application thereof in preparing a medicine or a health-care product with an anti-fatigue effect.

Background

Fatigue is also called fatigue, which is a subjective uncomfortable feeling of fatigue and weakness. It is meant that the body is unable to maintain a specific level of physiological activity or a predetermined level of exercise intensity during the physiological process. Fatigue is a common medical and neurological condition, and many diseases can cause fatigue, with different diseases causing different degrees of fatigue. Embodied in the nervous system, cardiovascular system and skeletal muscle.

According to the mechanism of fatigue occurrence, fatigue is mainly manifested in that: central fatigue, peripheral fatigue, spinal fatigue, synaptic fatigue, motor endplate fatigue and muscle fatigue. Fatigue is actually a protective response that prevents the body from developing life-threatening excessive failure. Fatigue is a comprehensive reaction of a plurality of physiological and biochemical changes in organisms, the pH value of muscle is reduced, blood sugar is reduced, glycogen is reduced, ATP and phosphocreatine (CP) in the brain are obviously reduced, gamma-aminobutyric acid (7-GABA) is obviously increased, the enzyme activity in tissues is reduced, and the like. These changes are both the result of the body's functional activities continuing to a certain extent and the factors that play an important role in the development of fatigue. If fatigue is not cleared in time, it will gradually accumulate, eventually leading to the dysfunction of the nervous, endocrine and immune system of the body, even organic lesions, which become important factors for destroying the physical and mental health of human beings. Depending on the location of fatigue and the metabolite factors, there are several hypotheses about fatigue: the theory of energy exhaustion, the theory of metabolite accumulation, the theory of oxygen-derived lipid peroxidation, the theory of endocrine dysfunction, and the theory of protective inhibition.

The fast-paced life, high-pressure academic, frequent overtime, serious sleep shortage and family pressure all cause great troubles to people. The upgrading of the industry optimizes social resource allocation, and simultaneously, the pressure of the whole society is increased invisibly, various fatigue symptoms are easy to be aroused, and various diseases can be caused if fatigue is not relieved in time. With the development of modern medicine, people can regulate the physiological state of the body by supplementing health care products and medicines with the anti-fatigue effect and relieve fatigue symptoms. General anti-fatigue drugs provide energy through the body, accelerate the clear rate of metabolites, regulate the internal environment of the body, increase the immunity of the body or directly stimulate the nervous system to achieve the anti-fatigue effect.

The probiotics is an active microorganism which is beneficial to a host and is formed by changing the flora at a certain part of the host by customizing in a human body, and the function of promoting nutrient absorption and keeping the intestinal tract healthy is realized by regulating the host mucous membrane and the system immune function or regulating the flora balance in the intestinal tract, so that a single microorganism which is beneficial to the host health or a mixed microorganism with a definite composition is generated. The anti-fatigue effect of the probiotics can be shown in the following steps: the probiotics have certain effect of enhancing the anti-fatigue capability of the human body, but the preparation of efficient anti-fatigue medicaments needs to be further researched.

According to the traditional Chinese medicine, the formation of fatigue is closely related to the dysfunction of the five internal organs of a human body, for example, the weakness of limbs is related to the spleen and stomach, the soreness of waist and the weakness of legs is related to the kidney, the deficiency of strength is related to the lung, the slurred head is related to the heart, and the intolerance of fatigue is related to the liver. Therefore, the ancient people refer to the spleen and stomach as the acquired root, the spleen and stomach are strong, the primordial qi is sufficient, the viscera function is normal, the anti-fatigue capability can be enhanced, and the immunity for preventing and resisting diseases is improved. The traditional Chinese medicine is used for conditioning organisms and enhancing the anti-fatigue capability, and is more and more favored by modern people.

Disclosure of Invention

In view of the above, the present invention firstly provides a new compound, and further research shows that the new compound has an anti-fatigue effect.

In addition, the invention also provides a traditional Chinese medicine probiotic fermentation product which also has an anti-fatigue effect.

In addition, the invention also provides a preparation method and application of the novel compound and a traditional Chinese medicine probiotic fermentation product.

The detailed technical scheme of the invention is as follows:

a compound of formula (I) or a pharmaceutically acceptable salt thereof;

the compound of formula (i) is named: methyl O-benzyl-N- (1H-indole-2-carbonyl) serinate.

Preferably, the pharmaceutically acceptable salt is a pharmaceutically acceptable acid addition salt, wherein the acid is selected from the group consisting of: citric acid, tartaric acid, lactic acid, acetic acid, succinic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, pyruvic acid, fumaric acid, maleic acid, hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, and sulfuric acid.

The present invention provides a process for the preparation of a compound of formula (i) which comprises the steps of:

(1) taking the traditional Chinese medicine raw materials, and fermenting with probiotics to obtain fermentation liquor;

(2) drying the fermentation liquor to obtain a crude product;

(3) separating the crude product by chromatography to obtain a compound of formula (I);

the traditional Chinese medicine raw materials in the step (1) are traditional Chinese medicine raw materials containing arillus longan, radix codonopsis, radix astragali, angelica sinensis, fructus lycii, honeysuckle and bighead atractylodes rhizome;

the probiotics in the step (1) are selected from two or more than two of bifidobacterium infantis, lactobacillus acidophilus, bifidobacterium bifidum and lactobacillus salivarius.

Preferably, the weight ratio of arillus longan, codonopsis pilosula, astragalus membranaceus, angelica sinensis, wolfberry fruits, honeysuckle flowers and bighead atractylodes rhizome in the traditional Chinese medicine raw materials in the step (1) is 1-5: 1-5: 1-5: 1-5: 1-5: 1-5: 1 to 5.

Most preferably, the weight ratio of arillus longan, radix codonopsis, radix astragali, angelica sinensis, fructus lycii, honeysuckle and bighead atractylodes rhizome in the traditional Chinese medicine raw materials in the step (1) is 1: 1: 1: 1: 1: 1: 1. namely, the weight and the dosage of the traditional Chinese medicine raw materials are equal.

Preferably, the specific method of step (1) is: extracting the Chinese medicinal materials with water, and concentrating the extractive solution to obtain concentrated solution; then adding a fermentation nutrient into the concentrated solution; and then adding probiotics for fermentation for 36-60 h under the conditions that the temperature is 37-42 ℃ and the pH is 6.5-7.5 to obtain fermentation liquor.

Further preferably, the amount of water used in the step (1) is 8-15 times of the amount of the traditional Chinese medicine raw materials.

Further preferably, the concentrated solution obtained in the step (1) is concentrated to 1/3-1/4 of the volume of the extracting solution.

Further preferably, the fermentation nutrient in step (1) comprises glucose 0.5% (weight/volume), yeast powder 0.1% (weight/volume), yeast extract 0.2% (weight/volume) and peptone 0.2% (weight/volume); the percentages are calculated on the volume of the concentrate.

Still more preferably, the nutrient for fermentation in step (1) further comprises 0.2% (w/v) potassium dihydrogen phosphate, 0.05% (w/v) magnesium sulfate, and 0.01% (w/v) ferrous sulfate; the percentages are calculated on the volume of the concentrate.

Even more preferably, said probiotic bacteria are selected from the group consisting of lactobacillus acidophilus and lactobacillus salivarius. The probiotics can be added in the form of dry powder, or can be activated by a conventional method and added in the form of bacteria liquid.

Preferably, the chromatographic technique described in step (3) is a preparative HPLC technique; the specific conditions of the preparative HPLC technique are as follows: adopting a reversed phase chromatographic column; selecting an ultraviolet detector, wherein the detection wavelength is 220-230 nm; using a trifluoroacetic acid aqueous solution with the volume percent of 0.05-0.2 as a mobile phase A, using a trifluoroacetic acid acetonitrile solution with the volume percent of 0.05-0.2 as a mobile phase B, wherein the mobile phase A: mobile phase B85: 15; and (3) collecting the part corresponding to the chromatographic peak for 8.0-9.0 min, and concentrating and drying to obtain the compound shown in the formula (I).

The invention also provides a traditional Chinese medicine probiotic fermentation product which comprises the compound shown in the formula (I).

The invention also provides a preparation method of the traditional Chinese medicine probiotic fermentation product, which comprises the following steps:

(1) taking the traditional Chinese medicine raw materials, and fermenting with probiotics to obtain fermentation liquor;

(2) drying the fermentation liquor to obtain the traditional Chinese medicine probiotic fermentation product;

the traditional Chinese medicine raw materials in the step (1) are traditional Chinese medicine raw materials containing arillus longan, radix codonopsis, radix astragali, angelica sinensis, fructus lycii, honeysuckle and bighead atractylodes rhizome;

the probiotics in the step (1) are selected from two or more than two of bifidobacterium infantis, lactobacillus acidophilus, bifidobacterium bifidum and lactobacillus salivarius.

Preferably, the weight ratio of arillus longan, codonopsis pilosula, astragalus membranaceus, angelica sinensis, wolfberry fruits, honeysuckle flowers and bighead atractylodes rhizome in the traditional Chinese medicine raw materials in the step (1) is 1-5: 1-5: 1-5: 1-5: 1-5: 1-5: 1 to 5.

Most preferably, the weight ratio of arillus longan, radix codonopsis, radix astragali, angelica sinensis, fructus lycii, honeysuckle and bighead atractylodes rhizome in the traditional Chinese medicine raw materials in the step (1) is 1: 1: 1: 1: 1: 1: 1. namely, the weight and the dosage of the traditional Chinese medicine raw materials are equal.

Preferably, the specific method of step (1) is: extracting the Chinese medicinal materials with water, and concentrating the extractive solution to obtain concentrated solution; then adding a fermentation nutrient into the concentrated solution; and then adding probiotics for fermentation for 36-60 h under the conditions that the temperature is 37-42 ℃ and the pH is 6.5-7.5 to obtain fermentation liquor.

Further preferably, the amount of water used in the step (1) is 8-15 times of the amount of the traditional Chinese medicine raw materials.

Further preferably, the concentrated solution obtained in the step (1) is concentrated to 1/3-1/4 of the volume of the extracting solution.

Further preferably, the fermentation nutrient in step (1) comprises glucose 0.5% (weight/volume), yeast powder 0.1% (weight/volume), yeast extract 0.2% (weight/volume) and peptone 0.2% (weight/volume); the percentages are calculated on the volume of the concentrate.

Still more preferably, the nutrient for fermentation in step (1) further comprises 0.2% (w/v) potassium dihydrogen phosphate, 0.05% (w/v) magnesium sulfate, and 0.01% (w/v) ferrous sulfate; the percentages are calculated on the volume of the concentrate.

Even more preferably, said probiotic bacteria are selected from the group consisting of lactobacillus acidophilus and lactobacillus salivarius. The probiotics can be added in the form of dry powder, or can be activated by a conventional method and added in the form of bacteria liquid.

The invention also provides application of the compound shown in the formula (I) or medicinal salt thereof or a traditional Chinese medicine probiotic fermentation product in preparation of medicines or health-care products.

Preferably, the medicament is a medicament with an anti-fatigue effect; the health care product has the function of resisting fatigue.

Preferably, the medicine or health care product contains a compound shown in the formula (I) or a medicinal salt thereof or a traditional Chinese medicine probiotic fermentation product and a carrier; the mass fraction of the compound of formula (I) or the medicinal salt thereof or the traditional Chinese medicine probiotic fermentation product is 5-95%.

Further preferably, the carrier comprises at least one of a solvent, a polymer and a liposome. Still further preferably, the solvent includes, but is not limited to, water, physiological saline, and other non-aqueous solvents. Still further preferably, the polymer includes, but is not limited to, polylysine, polyethyleneimine and modifications thereof, chitosan, polylactic acid, gelatin. Still further preferably, the liposome may be, but is not limited to, cholesterol, soy lecithin, egg yolk lecithin.

Even more preferably, the carrier further comprises one or more of a diluent and an excipient. Specifically, the diluent includes one or more of starches, sugars, celluloses and inorganic salts. Specifically, the excipient comprises one or more of a binder, a filler, a lubricant in a tablet, a matrix part in a semisolid preparation ointment or cream, a preservative in a liquid preparation, an antioxidant, a flavoring agent, an aromatic agent, a cosolvent, an emulsifier and a coloring agent.

Further preferably, the mass fraction of the compound of formula (I) or the pharmaceutically acceptable salt thereof or the traditional Chinese medicine probiotic fermentation product is 10-90%, 15-85% or 30-80%.

Preferably, the health product or the medicine is in the form of tablet, capsule, powder, granule, pill, syrup, solution, suspension or aerosol.

Preferably, the health product is in the form of gel or water.

Further preferably, the health product further comprises an auxiliary material matrix, wherein the auxiliary material matrix comprises one or more of monosaccharide, oligosaccharide, polysaccharide, amino acid, preservative and pH regulator.

Has the advantages that: (1) the invention provides a brand-new compound shown in formula (I), a traditional Chinese medicine probiotic fermentation product and a preparation method thereof; the compound of the formula (I) and the traditional Chinese medicine probiotic fermentation product have excellent anti-fatigue effect; can be used for preparing medicines and health products.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only drawings of some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 shows the mass spectrum of a compound of formula (I) according to the invention.

FIG. 2 shows a hydrogen spectrum of a compound of formula (I) according to the invention.

FIG. 3 shows the carbon spectrum of the compound of formula (I) according to the invention.

FIG. 4 shows the effect of probiotic fermentation product of Chinese herbs and monomer compound of formula (I) on the swimming time of mice.

FIG. 5 shows the effect of probiotic fermented product of Chinese medicinal materials and monomer compound with formula (I) on liver glycogen and muscle glycogen of mouse.

FIG. 6 shows the effect of probiotic fermented product of Chinese medicinal materials and monomer compound of formula (I) on blood lactate and blood lactate dehydrogenase of mouse.

FIG. 7 shows the effect of probiotic fermentation products of Chinese herbs and monomeric compounds of formula (I) on mouse serum urea nitrogen.

Detailed Description

The technical solution of the present invention will be clearly and completely described with reference to the following examples. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

EXAMPLE 1 preparation of the Compound of formula (I)

(1) Extracting the traditional Chinese medicine raw materials with 8 times of water, soaking for 50 minutes, and performing ultrasonic extraction for 30 minutes; concentrating the extractive solution to 1/3 volume to obtain concentrated solution; then adding a fermentation nutrient into the concentrated solution; then adding probiotics, and fermenting for 48h under the conditions that the temperature is 39 ℃ and the pH value is 7.0 to obtain fermentation liquor;

(2) centrifuging the fermentation liquor at 8000r/min for 30min, removing precipitate, retaining supernatant, and freeze drying the supernatant to obtain crude product;

(3) separating the crude product by chromatography to obtain a compound of formula (I);

the traditional Chinese medicine raw materials in the step (1) are prepared from arillus longan, codonopsis pilosula, astragalus membranaceus, angelica sinensis, wolfberry fruits, honeysuckle and bighead atractylodes rhizome according to a weight ratio of 1: 1: 1: 1: 1: 1: 1 (namely the usage of the traditional Chinese medicine raw materials is equal);

the addition amount of the fermentation nutrient in the step (1) is calculated according to the volume of the concentrated solution: it comprises glucose 0.5% (weight/volume), yeast powder 0.1% (weight/volume), yeast extract 0.2% (weight/volume) and peptone 0.2% (weight/volume); potassium dihydrogen phosphate 0.2% (w/v), magnesium sulfate 0.05% (w/v), ferrous sulfate 0.01% (w/v);

the probiotics in the step (1) are lactobacillus acidophilus dry powder and lactobacillus salivarius dry powder; according to the volume of the concentrated solution, the addition amount of the lactobacillus acidophilus dry powder is 7% (weight/volume), and the addition amount of the lactobacillus salivarius dry powder is 3% (weight/volume);

the chromatographic technique in the step (3) is a preparative HPLC technique; the specific conditions of the preparative HPLC technique are as follows: adopting an ODS (oxide dispersion strengthened) reversed-phase chromatographic column; selecting an ultraviolet detector, wherein the detection wavelength is 220 nm; using a 0.1 volume percent trifluoroacetic acid aqueous solution as a mobile phase A, using a 0.1 volume percent trifluoroacetic acid acetonitrile solution as a mobile phase B, wherein the ratio of the mobile phase A: mobile phase B85: 15; and (3) collecting the part corresponding to the chromatographic peak for 8.0-9.0 min, and concentrating and drying to obtain the compound shown in the formula (I).

The mass spectrum, hydrogen spectrum and carbon spectrum data of the compound of the formula (I) are as follows (the specific spectrogram is shown in figures 1-3):

mass spectrometry data showed 375.1 to be [ M + Na ]]⁺Molecular formula C₂₀H₂₀N₂O₄；¹H NMR(600MHz,DMSO)δ:11.64(d,1H),8.82(d,1H),7.64(dd,1H),7.43(dd,1H),7.32(m,4H),7.28(td,1H),7.20(td,1H),7.05(td,1H),4.82(td,1H),4.55(td,2H),3.82(m,2H),3.67(s,3H).¹³C NMR(151MHz,DMSO)δ:170.64,161.16,137.97,136.56,130.82,128.26,127.54,126.98,123.58,121.65,119.79,112.32,103.79,72.09,68.79,52.53,52.08,38.24.

The Methyl O-benzyl-N- (1H-indole-2-carbonyl) serinate of the compound of the formula (I) can be confirmed to be prepared by the method through mass spectrum, hydrogen spectrum and carbon spectrum analysis.

Example 2 preparation of traditional Chinese medicine probiotic fermentation product

(1) Extracting the traditional Chinese medicine raw materials with 8 times of water, soaking for 50 minutes, and performing ultrasonic extraction for 30 minutes; concentrating the extractive solution to 1/3 volume to obtain concentrated solution; then adding a fermentation nutrient into the concentrated solution; then adding probiotics, and fermenting for 48h under the conditions that the temperature is 39 ℃ and the pH value is 7.0 to obtain fermentation liquor;

(2) centrifuging the fermentation liquor at 8000r/min for 30min, removing precipitate, retaining supernatant, and freeze drying the supernatant to obtain traditional Chinese medicine probiotic fermentation product;

the traditional Chinese medicine raw materials in the step (1) are prepared from arillus longan, codonopsis pilosula, astragalus membranaceus, angelica sinensis, wolfberry fruits, honeysuckle and bighead atractylodes rhizome according to a weight ratio of 1: 1: 1: 1: 1: 1: 1 (namely the usage of the traditional Chinese medicine raw materials is equal);

the addition amount of the fermentation nutrient in the step (1) is calculated according to the volume of the concentrated solution: it comprises glucose 0.5% (weight/volume), yeast powder 0.1% (weight/volume), yeast extract 0.2% (weight/volume) and peptone 0.2% (weight/volume); potassium dihydrogen phosphate 0.2% (w/v), magnesium sulfate 0.05% (w/v), ferrous sulfate 0.01% (w/v);

the probiotics in the step (1) are lactobacillus acidophilus dry powder and lactobacillus salivarius dry powder; according to the volume of the concentrated solution, the addition amount of the lactobacillus acidophilus dry powder is 7% (weight/volume), and the addition amount of the lactobacillus salivarius dry powder is 3% (weight/volume).

Comparative example 1 Chinese medicinal extract

Extracting the traditional Chinese medicine raw materials with 8 times of water, soaking for 50 minutes, and performing ultrasonic extraction for 30 minutes; concentrating and drying the extract to obtain the traditional Chinese medicine extract. The traditional Chinese medicine raw materials comprise arillus longan, codonopsis pilosula, astragalus membranaceus, angelica sinensis, wolfberry, honeysuckle and bighead atractylodes rhizome according to a weight ratio of 1: 1: 1: 1: 1: 1: 1 (namely the dosage of the traditional Chinese medicine raw materials is equal).

Experimental example 1 study on anti-fatigue effect of probiotic fermented product of traditional Chinese medicine and monomeric compound having structure of formula (I)

(1) Grouping and feeding animals: after 18-20g Kunming male mice are adapted for 7 days under the experimental environment, the mice are randomly divided into a blank control group (0) (the same amount of normal saline is given every day) and a 400mg/kg/d traditional Chinese medicine extract group (ZY) prepared in the comparative example 1, a traditional Chinese medicine probiotic fermentation product group (ZYD) prepared in the example 2 and a monomer compound group (ZYDD) with the structure of the formula (I). Each group of 10 mice was gavaged at 8:00 am daily with a gavage volume of 0.5mL for 21 days with free intake and drinking during gavage.

(2) Weight bearing swimming experiment

After the last gastric lavage of the mice for 0.5h, the mice were loaded with a 3% weight in a 6cm long string 1cm from the tip of the tail. The mice are placed in a swimming box with the water depth of 30cm and the water temperature of 25.0 +/-1.0 ℃ for swimming, and the time from the beginning of swimming to the bottom sinking of the mice is recorded and the time of the mouse weight swimming is recorded. The direct expression of the improvement of the exercise endurance is the enhancement of the anti-fatigue capability, and the length of the swimming time can better reflect the anti-fatigue degree of the animal exercise.

The effect of the probiotic metabolites of the traditional Chinese medicine on the swimming time of mice is shown in figure 4. The swimming time of the traditional Chinese medicine extract group, the traditional Chinese medicine probiotic fermentation product group and the monomer compound group with the structure of the formula (I) is respectively increased by 57.2s, 195.4s and 237.0s compared with that of the blank control group. The method shows that the traditional Chinese medicine probiotic fermentation product and the monomer compound with the structure of the formula (I) have certain effects on prolonging the swimming time of the mouse; wherein the monomer compound with the structure of formula (I) has the best effect; and are all stronger than the traditional Chinese medicine extract; this shows that the traditional Chinese medicine probiotic fermentation product obtained by fermenting the traditional Chinese medicine extract by the method of the invention and the monomeric compound with the structure of formula (I) have more excellent anti-fatigue effect.

(3) Liver glycogen and muscle glycogen assay

After the mice are subjected to gastric lavage for 0.5h for the last time, immediately killing the mice, taking the muscles of the liver and the hind legs, rinsing the muscles with normal saline, drying the muscles with filter paper, accurately weighing 100mg of the liver (the muscles of the hind legs), adding 8mL of TCA, homogenizing for 1min, pouring the homogenate into a centrifuge tube, centrifuging for 15min at 3000r/min, taking 1mL of supernatant, adding 5mL of 95% ethanol, fully and uniformly mixing until no interface exists between the two liquids, plugging the plug, and standing at room temperature overnight. Centrifuge at 3000r/min for 15min on day 2. The supernatant was decanted and the tube was placed upside down for 10 min. Glycogen was dissolved in 2mL of distilled water.

Reagent blank tube: draw 2mL of distilled water into a clean centrifuge tube standard: 0.5mL of glucose standard solution (1mg/mL) and 1.5mL of distilled water were aspirated into the same tube. Add 10mL anthrone reagent into each tube, boil for 15min, move to ice water bath, cool, and measure absorbance at 620nm wavelength after zeroing with reagent blank tube. Calculated as follows:

liver (muscle) glycogen (mg/100g liver (muscle) tissue) ═ i/ii × 0.5 × (v/g) × 100 × 0.9 where i is sample tube absorbance; v is the volume of extract (mL); II is the absorbance of a standard tube; 0.5 is the glucose content in 0.5mL of glucose standard solution; g is the weight of liver (muscle) tissue (g); 0.9 is a coefficient for converting glucose into glycogen.

Energy exhaustion is one of the important causes of fatigue generation, glycogen is an important energy substance in the body, and exhaustion of the reserve is one of the main causes of fatigue generation and is also a decisive factor of the early and late occurrence of fatigue. The influence result of the traditional Chinese medicine probiotic fermentation product and the monomeric compound with the structure of the formula (I) on the content of the mouse liver glycogen is shown in figure 5, and the experimental result shows that: compared with a control group, after 21 days, the storage of liver and muscle glycogen of the mice is increased to a certain extent by using the fermentation product of the traditional Chinese medicine probiotics, and after 21 days, the storage of liver and muscle glycogen of the mice is increased most obviously compared with that of a blank group by using the monomeric compound with the structure of formula (I) and the metabolism product of the traditional Chinese medicine probiotics. The results show that the traditional Chinese medicine probiotic fermentation product and the monomeric compound with the structure of the formula (I) can increase the reserves of liver and muscle glycogen of the experimental mice, provide more energy substances for the body and further delay the generation of fatigue.

(4) Blood lactate and blood lactate dehydrogenase Activity measurement

After the test object is given to the mouse for 0.5h at the last time, 20 mu L of blood is collected by a capillary glass tube eyeball, and 50 mu L of the blood is added into the membrane breaking liquid to uniformly break the cells by oscillation. Then swimming the mouse in water with the temperature of 30.0 +/-0.5 ℃ for 10min, stopping, immediately taking 20 mu L of blood, adding into 50 mu L of membrane breaking liquid, and uniformly oscillating. After resting for 20min, collecting blood again by 20 μ L, adding into 40 μ L of membrane-breaking liquid, and shaking uniformly. Measuring with lactic acid analyzer, and multiplying the obtained measurement value by 3 to obtain blood lactic acid value (mmol/L), wherein the blood lactic acid dehydrogenase activity is performed according to the requirements of the kit.

The accumulation degree of lactic acid in the body depends on the speed of lactic acid production and removal, and the speed of lactic acid removal is high, which means that fatigue can be rapidly eliminated. After strenuous exercise, lactate dehydrogenase in the animal body can be increased to accelerate the elimination of blood lactate, thereby reducing the fatigue of the body of the exercise animal. The influence of the traditional Chinese medicine probiotic fermentation product and the monomer compound with the structure of the formula (I) on the LDH activity of the mice is shown in figure 6, and compared with a blank control group, the serum lactate dehydrogenase activity (LDH) of the experimental mice of the traditional Chinese medicine probiotic fermentation product group and the monomer compound group with the structure of the formula (I) is obviously improved after exercise. Compared with a blank control group, the serum lactate dehydrogenase activity (LDH) of the mice in the traditional Chinese medicine extract group, the traditional Chinese medicine probiotic fermentation product group and the monomer compound group with the structure of the formula (I) are respectively increased by 0.95%, 11.7% and 15.8%, which shows that the traditional Chinese medicine probiotic fermentation product prepared on the basis of the traditional Chinese medicine extract and the monomer compound with the structure of the formula (I) can improve the level of the serum lactate dehydrogenase activity (LDH) of the mice after exercise.

(5) Serum urea nitrogen

The mice fasting for 12h, and the loading mode is the same after the last gavage for 30 min. Swimming in water at 25 + -0.5 deg.C for 10min, resting for 20min, collecting blood in orbit, centrifuging at 4 deg.C and 6000r/min for 10min to separate serum, storing at-20 deg.C for testing, and testing urea nitrogen according to the kit.

The poorer the adaptability of the organism to the load, the more obvious the increase of the urea nitrogen in the body is, and the urea nitrogen content of the organism is increased along with the increase of the exercise load, so the urea nitrogen is an ideal and sensitive index for detecting the fatigue of the organism. The result of analyzing the influence of the traditional Chinese medicine probiotic fermentation product and the monomer compound with the structure shown in the formula (I) on the urea nitrogen content in the serum of the mouse can be known (see figure 7), and the traditional Chinese medicine probiotic fermentation product and the monomer compound with the structure shown in the formula (I) can well reduce the urea nitrogen content in the serum of the mouse. The traditional Chinese medicine probiotic fermentation product and the monomer compound with the structure shown in the formula (I) can enhance the effect of mice on removing urea nitrogen generated in movement and reduce the content of urea nitrogen, wherein the effect of enhancing the effect of mice on removing urea nitrogen generated in movement is most obvious when the monomer compound with the structure shown in the formula (I) is used.

The experiments show that the traditional Chinese medicine probiotic fermentation product and the monomer compound with the structure of the formula (I) have excellent anti-fatigue effect; in particular to the separated monomer compound with the structure of formula (I), which has stronger anti-fatigue effect.

While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.