CN112521447A

CN112521447A - Compound, traditional Chinese medicine probiotic fermentation product and application of compound and traditional Chinese medicine probiotic fermentation product in preparation of medicine or health-care product with anti-aging effect

Info

Publication number: CN112521447A
Application number: CN201911170311.1A
Authority: CN
Inventors: 刘志刚; 刘杰; 刘晓宇
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-11-26
Filing date: 2019-11-26
Publication date: 2021-03-19

Abstract

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-aging 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 comprising purple perilla, astragalus, lily, rhodiola rosea, glossy privet fruit, pseudo-ginseng, ginkgo leaf, mint and gorgon fruit. The compound shown in the formula (I) and the traditional Chinese medicine probiotic fermentation product have excellent effects of resisting oxidation and delaying senescence; can be used for preparing medicines and health products.

Description

Compound, traditional Chinese medicine probiotic fermentation product and application of compound and traditional Chinese medicine probiotic fermentation product in preparation of medicine or health-care product with anti-aging effect

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 preparation of a medicine or a health-care product with an anti-aging effect.

Background

As a person ages, there are aging conditions such as white hair, tooth loss, muscular atrophy, angiosclerosis, slow sensory response, memory deterioration, and metabolic function decline. The phenomenon of irreversible functional decline and gradual death of various organs, tissues and cells in the body accompanying the increase of age is generally called aging of the body. The aging of the group has the cellular biology basis. During aging, cells of tissues and organs also undergo a gradual decline in morphological structure and physiological function. With the increase of age, the self-renewal and proliferation and differentiation abilities of cells are reduced, so that the damage of tissues and organs is difficult to repair, the normal physiological function is difficult to maintain, and the aging of the organism inevitably occurs. The problem of delaying aging needs to be explored and researched.

The understanding of the aging mechanism by the basic theory of traditional Chinese medicine is centered on the zang-fu organs, and the kidneys play an important role in the aging mechanism of traditional Chinese medicine. The selection of the antioxidant and anti-aging traditional Chinese medicines (including single traditional Chinese medicine, compound preparation and traditional Chinese medicine extract) not only accords with the comprehensive, early and long-term principles, but also depends on the actual condition of an individual and is in accordance with the condition of people. More than 2000 years ago, the earliest classic traditional Chinese medicine book, the "internal classic" in China, had records on the aging process of human beings. The records of Ling Shu and Tian Nian Ling are: for example, liver qi begins to decline in the fifty years old, liver lobes begin to thin, bile begins to decline, and the eyes begin to be unknown. Sixty years old, heart-qi begins to decline, so the patient feels good lying because of the sorrow of the heart-qi and the exhaustion of blood-qi. Seventy years old, spleen qi deficiency and dry skin. Eighty years old, the lung qi is too weak, the body is too strong and the body is too weak. Ninety years old, kidney qi scorching, and four zang organs with empty vessels.

The understanding of the aging mechanism by the basic theory of traditional Chinese medicine takes viscera as the core, and comprises theories of kidney deficiency aging, liver depression aging, spleen and stomach weakness aging, qi stagnation, blood stasis, phlegm turbidity aging and the like. The traditional Chinese medicine considers that the aging is mainly caused by kidney deficiency, spleen deficiency and qi and blood deficiency. Therefore, tonifying the kidney, strengthening the spleen and replenishing qi are basic ways of delaying senescence, and activating blood circulation to dissipate blood stasis is a main method of delaying senescence. Some Chinese medicines with the functions of invigorating vital energy, strengthening body resistance, consolidating constitution, tonifying and the like can achieve the aim of resisting aging by the principles of antioxidation and the like.

Lily is sweet and neutral in nature. It enters spleen, lung and heart meridians. The effect is as follows: tonify primordial qi, recover pulse to stop collapse, tonify spleen and lung, promote fluid production, and induce tranquilization. The main treatment is as follows: can be used for treating asthenia, collapse, cold limbs, weak pulse, spleen deficiency, anorexia, loose stool, short breath, asthenia, lung deficiency, asthma, cough due to body fluid deficiency, internal heat, diabetes, asthenia, palpitation, insomnia, sexual impotence, and cold womb; heart failure, cardiogenic shock. Has special effect on central nervous system, can balance brain excitation and inhibition, enhance physical strength, has remarkable antifatigue effect, and can improve mood and sleep condition, and eliminate general weakness and headache.

Mint, slightly sweet and warm in nature. It enters liver and kidney meridians. The effect is as follows: dispel wind-damp, tonify liver and kidney, strengthen tendons and bones, activate blood. The main treatment is as follows: arthralgia due to wind-cold-dampness, lumbago, gonalgia, flaccidity of bones and muscles, retardation of small children, weakness, traumatic injury, fracture, edema, beriberi, and pruritus vulvae. Is used for treating deficiency of liver and kidney, enhancing the resistance of the body to harmful stimulation factors, has good anti-fatigue effect, and can obviously improve the hypoxia tolerance and enhance the nonspecific immunity function of the body.

Gorgon fruit, sweet and warm in nature. It enters spleen and lung meridians. The effect is as follows: benefiting wei-defensive qi, consolidating superficial resistance, invigorating qi, invigorating yang, expelling toxin, promoting granulation, inducing diuresis, and relieving swelling. The main treatment is as follows: can be used for treating deficiency of qi, asthenia, anorexia, loose stool, collapse of middle-warmer energy, chronic diarrhea, rectocele, spontaneous perspiration, night sweat, blood deficiency, sallow complexion, dorsal furuncle, edema, internal heat, and diabetes. Has effects in invigorating qi, invigorating yang, consolidating superficial resistance, arresting sweating, expelling pus, inducing diuresis, relieving swelling, enhancing immunity, promoting metabolism, lowering blood pressure, promoting urination, and inhibiting bacteria.

Ginkgo, semen Ginkgo, is sweet and neutral in nature. Has little toxicity. It enters heart and lung meridians. The effect is as follows: promoting blood circulation, removing blood stasis, dredging collaterals, relieving pain, astringing lung, relieving asthma, eliminating turbid pathogen, and reducing blood lipid. The main treatment is as follows: can be used for treating blood stasis, obstruction of collaterals, thoracic obstruction, cardialgia, apoplexy, hemiplegia, cough with asthma due to lung deficiency, and hyperlipidemia. As a traditional Chinese medicinal material, the Chinese medicinal composition is known for rejuvenation of the brain. Ginkgo is also a highly effective antioxidant. Experiments show that the gingko has higher efficiency in scavenging free radicals than vitamin E, and can effectively prevent high-fat cell membranes from being oxidized. Ginkgo biloba can restore the integrity of the cell membrane after the cells are destroyed by free radicals. In addition, ginkgo biloba may restore the ability of brain cells to receive signals from the nervous conduction system that direct the brain to perform work.

Other Chinese medicinal materials such as radix rehmanniae Preparata, radix asparagi, radix Ophiopogonis, radix Angelicae sinensis, cortex moutan, Notoginseng radix, radix Codonopsis, Atractylodis rhizoma, Ganoderma, fructus crataegi, Polygoni Multiflori radix, Poria, and rhizoma Polygonati can be used as antiaging agent. At present, a few reports exist on preparing lily, mint, ginkgo and gordon euryale seed into beverages or preparations. Although the methods have certain innovation, the traditional extraction and preparation methods are still adopted, so that the absorption and utilization rate of the human body is low, and certain limitations exist. The anti-aging drugs should not be indiscriminately used, and the application of the anti-aging drugs should meet the comprehensive, early and long-term principles, and the anti-aging drugs should be selected according to the individual's conditions from the actual point of view. Delaying aging is one of the key points and difficulties in the current life science research. Therefore, the development of antioxidant and anti-aging medicaments with independent intellectual property rights also becomes a hot point for the research and development of traditional Chinese medicines.

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 antioxidant effect and anti-aging effect.

In addition, the invention also provides a traditional Chinese medicine probiotic fermentation product which also has the effects of resisting oxidation and delaying senescence.

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:

Benzyl O-benzyl-N-(2,4-dimethoxybenzoyl)serylglycinate。

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 comprising purple perilla, astragalus, lily, rhodiola rosea, glossy privet fruit, pseudo-ginseng, ginkgo leaf, mint and gorgon fruit;

the probiotics in the step (1) are selected from one or a mixture of more than two of bifidobacteria, lactobacillus liquid, bifidobacterium adolescentis or bifidobacterium infantis.

Preferably, the weight ratio of the perilla, the astragalus, the lily, the rhodiola rosea, the glossy privet fruit, the pseudo-ginseng, the ginkgo leaf, the mint and the gordon euryale seed in the traditional Chinese medicine raw materials in the step (1) is 1-5: 1-5: 1-5: 1-5: 1-5: 1-5: 1-5: 1-5: 1 to 5.

Most preferably, the weight ratio of the perilla, the astragalus, the lily, the rhodiola rosea, the glossy privet fruit, the pseudo-ginseng, the ginkgo leaf, the mint and the gordon euryale seed in the traditional Chinese medicine raw materials in the step (1) is 1: 1: 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 to ferment for 24-48 h under the conditions that the temperature is 35-40 ℃ 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.

The probiotic is a combination of one selected from bifidobacterium and lactobacillus and one selected from bifidobacterium adolescentis and bifidobacterium infantis. 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 0.1-0.3 as a mobile phase A, using a trifluoroacetic acid acetonitrile solution with the volume percent of 0.1-0.3 as a mobile phase B, wherein the mobile phase A: mobile phase B-78: 22; and (3) collecting the part corresponding to the chromatographic peak for 13.0-13.7 min, 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:

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

The invention also provides application of the compound of 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 has an anti-oxidation effect and/or an anti-aging effect; the health care product has an anti-oxidation effect and/or an anti-aging effect.

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 effects of resisting oxidation and delaying senescence; can be used for preparing medicines and health products; (2) experiments show that the traditional Chinese medicine probiotic metabolite can obviously improve oxidative damage caused by D-galactose induction, obviously improve the activities of SOD, GSH-Px and T-AOC in mouse serum and liver, reduce the MDA content of lipid peroxides in mouse serum and liver, and improve the antioxidant capacity of mice.

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 is a graph showing the measurement of the activity of probiotic fermented products of Chinese herbs and monomeric compounds having the structure of formula (I) on mouse serum GSH-Px.

FIG. 5 is a graph showing the measurement of the T-AOC activity of the fermented product of probiotic bacteria of Chinese traditional medicine and the monomeric compound with the structure of formula (I) on mouse serum.

FIG. 6 shows the measurement of SOD activity of mouse serum by probiotic fermented product of Chinese medicinal materials and monomer compound with formula (I).

FIG. 7 is a graph of the measurement of the mouse serum MDA content of the traditional Chinese medicine probiotic fermentation product and the monomeric compound with the structure of formula (I).

FIG. 8 is a graph showing the measurement of the activity of probiotic fermented products of Chinese herbs and monomeric compounds of formula (I) on mouse liver GSH-Px.

FIG. 9 is a graph showing the measurement of the T-AOC activity of the traditional Chinese medicine probiotic fermentation product and the monomeric compound with the structure of formula (I) on mouse liver.

FIG. 10 shows the measurement of SOD activity of mouse liver by fermented product of probiotic bacteria of Chinese medicinal materials and monomer compound of formula (I).

FIG. 11 is a graph of the measurement of the traditional Chinese medicine probiotic fermentation product and the monomeric compound with the structure of formula (I) on the MDA content of the mouse liver.

FIG. 12 shows the effect of probiotic fermentation of Chinese herbs and the monomeric compound of formula (I) on the longevity of N2 C.elegans.

FIG. 13 effect of probiotic metabolites of traditional Chinese medicine on the survival rate of N2 C.elegans under oxidative stress conditions.

FIG. 14 Effect of traditional Chinese medicine probiotic metabolites on N2 C.elegans ROS content under oxidative stress conditions.

FIG. 15 effect of probiotic metabolites of traditional Chinese medicine on MDA activity of N2 caenorhabditis elegans under oxidative stress conditions.

FIG. 16 the effect of probiotic metabolites of traditional Chinese medicine on the SOD activity of N2 C.elegans under oxidative stress conditions.

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 10 times of water, soaking for 60 minutes, and performing ultrasonic extraction for 60 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 36h under the conditions that the temperature is 37 ℃ 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;

the traditional Chinese medicine raw materials in the step (1) are prepared from perilla, astragalus, lily, rhodiola rosea, glossy privet fruit, pseudo-ginseng, ginkgo leaf, mint and gordon euryale seed according to a weight ratio of 1: 1: 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 dry powder and bifidobacterium infantis dry powder; calculated according to the volume of the concentrated solution, the addition amount of the lactobacillus dry powder is 5 percent (weight/volume), and the addition amount of the bifidobacterium infantis dry powder is 2 percent (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 0.2 volume percent trifluoroacetic acid aqueous solution as a mobile phase A, using a 0.2 volume percent trifluoroacetic acid acetonitrile solution as a mobile phase B, wherein the ratio of the mobile phase A: mobile phase B-78: 22; and (3) collecting the part corresponding to the chromatographic peak for 13.0-13.7 min, 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 show [ M + Na [)]⁺529.2, molecular formula C₂₈H₃₀N₂O₇,¹H NMR(500MHz,DMSO)δ:8.64(t,1H),8.55(d,1H),7.92(d,1H),7.31(m,10H),6.68(m,2H),5.12(s,2H),4.75(m,1H),4.51(s,2H),3.95(d,2H),3.56(m,2H),3.37(s,6H),3.07(m,1H).¹³C NMR(126MHz,DMSO)δ:170.26,169.52,163.68,163.25,158.98,138.16,135.87,132.83,128.40,128.21,128.05,127.91,127.44,127.40,113.69,105.97,98.68,72.13,70.09,65.88,56.25,55.57,52.82,40.94.

The Benzyl O-Benzyl-N- (2, 4-dimethoxybenzyl) seriylgylcetate prepared by the method can be confirmed to be obtained by mass spectrum, hydrogen spectrum and carbon spectrum analysis.

Example 2 preparation of traditional Chinese medicine probiotic fermentation product

(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 probiotics in the step (1) are lactobacillus dry powder and bifidobacterium infantis dry powder; calculated by the volume of the concentrated solution, the addition amount of the lactobacillus dry powder is 5 percent (weight/volume), and the addition amount of the bifidobacterium infantis dry powder is 2 percent (weight/volume).

Comparative example 1 Chinese medicinal extract

Extracting the traditional Chinese medicine raw materials with 10 times of water, soaking for 60 minutes, and performing ultrasonic extraction for 60 minutes; concentrating and drying the extract to obtain the traditional Chinese medicine extract. The traditional Chinese medicine raw materials comprise perilla, astragalus, lily, rhodiola rosea, glossy privet fruit, pseudo-ginseng, ginkgo leaves, mint and gordon euryale seed according to a weight ratio of 1: 1: 1: 1: 1: 1: 1: 1: 1 (namely the dosage of the traditional Chinese medicine raw materials is equal).

Experimental example 1 experiment of oxidative damage of mice induced by D-galactose caused by fermented product of probiotic bacteria of Chinese medicinal herbs and monomeric compound having structure of formula (I)

(1) Grouping and feeding animals

Taking 70 healthy mice, feeding at 18-22 deg.C under natural illumination, and feeding with common feed for 5 d. Randomly dividing mice into 5 groups, (a normal Control group (Control), a model Control group (DG), a traditional Chinese medicine extract group (TCM) prepared in a comparative example 1, a traditional Chinese medicine probiotic fermentation product group (TCMPM) prepared in an example 2 and a monomer compound group (TPMSC) with a structure of a formula (I), performing abdominal injection of 700 mg/(kg.d) D-galactose on the rest groups except the normal Control group to establish an oxidative damage model, performing intragastric perfusion on 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) according to the dose of 200 mg/(kg.d) to obtain corresponding compounds, performing injection of physiological saline with the same volume to obtain distilled water with the same volume to perform intragastric perfusion for 1 time by weighing 2D, continuously taking 28D, taking blood from eyeballs to kill the mice, and measuring the superoxide dismutase (SOD) activity in serum and livers, Glutathione peroxidase (GSH-Px) activity, Malondialdehyde (MDA) content and total antioxidant capacity (T-AOC), and the in vivo antioxidant activity of traditional Chinese medicine extract, traditional Chinese medicine probiotic fermentation product and monomer compound with formula (I) structure is studied.

(2) Sample index detection

After the mice are subjected to last 1d gastric lavage, fasting is carried out for 12h, blood is taken from eye sockets, centrifugation is carried out for 20min at 5000r/min, serum is separated, supernatant is taken, and the MDA content, GSH-Px, SOD and T-AOC activity of the serum are detected according to the kit instructions. Dissecting a mouse, quickly taking out the liver, washing with normal saline, removing water by using absorbent paper, weighing 0.5g of liver tissue, placing the liver tissue in a homogenizer, adding 10 times volume of precooled normal saline to prepare 10% liver tissue homogenate, centrifuging at 4000r/min for 15min, taking supernatant, and determining GSH-Px, T-AOC, SOD activity and MDA content of a liver tissue sample according to a kit specification.

(3) Results of the experiment

From FIGS. 4, 5, 6 and 7, it is clear that after D-galactose injection, the GSH-Px, SOD and T-AOC activities in the serum of the mice were significantly decreased and the MDA content was significantly increased compared to the normal control group. After the traditional Chinese medicine extract, the traditional Chinese medicine probiotic fermentation product and the monomer compound with the structure of the formula (I) are perfused, the GSH-Px and T-AOC activities of the mouse serum are remarkably increased compared with those of a model injury group; compared with a model injury group, after gastric lavage of traditional Chinese medicine extract, traditional Chinese medicine probiotic fermentation product and monomer compound with the structure of formula (I), the activity of SOD is obviously improved; after the traditional Chinese medicine extract, the traditional Chinese medicine probiotic fermentation product and the monomeric compound with the structure of the formula (I) are perfused, compared with a model injury group, the MDA content is obviously reduced; wherein the activity of the monomer compound SOD with the structure of formula (I) is 1.26 times of that of the model group, the activity of GSP-Px is 1.36 times of that of the model group, the activity of T-AOC is 1.61 times of that of the model group, and the MDA content is 69.6% of that of the model group.

As can be seen from fig. 8, 9, 10, and 11, after D-galactose injection, the mouse liver had a significant decrease in GSH-Px activity, a significant decrease in T-AOC activity, and a significant increase in MDA content, as compared to the model injury group, as compared to the SOD activity and the normal control group; the T-AOC activity of mouse liver in the traditional Chinese medicine probiotic fermentation product group and the monomer compound group with the structure of formula (I) is obviously improved compared with that of a model injury group, but is still lower than that of a normal control group after the experiment is finished; compared with the model injury group, the activity of GSH-Px and SOD of mouse liver in the traditional Chinese medicine probiotic fermentation product group and the monomer compound group with the structure of formula (I) is obviously improved; compared with the model injury group, the traditional Chinese medicine probiotic fermentation product group and the monomer compound group with the structure of the formula (I) obviously reduce the MDA content of the liver of the mouse; in the monomer compound group with the structure of the formula (I), the activity of SOD is 1.73 times that of the model group, the activity of GSP-Px is 1.31 times that of the model group, the activity of T-AOC is increased by 1.74 times that of the model group, and the content of MDA is 69.1% that of the model group.

The experiment shows that the antioxidant effect of the traditional Chinese medicine probiotic fermentation product and the monomeric compound with the structure of the formula (I) is greater than that of a simple traditional Chinese medicine extract; the traditional Chinese medicine probiotic fermentation product prepared by the method and the monomeric compound with the structure of the formula (I) have more excellent antioxidant effect on the basis of the traditional Chinese medicine extract; in particular to the monomer compound with the structure of formula (I) which is obtained by separation and has stronger antioxidation.

Experimental example 2 evaluation of anti-aging Effect of fermented product of Chinese medicinal probiotic and monomeric compound having structure of formula (I)

(1) The wild type caenorhabditis elegans N2 is cultured at 20 ℃, and randomly divided into 4 groups (the number of each group is not less than 60), wherein each group comprises 3 parallel Control groups, namely a normal Control group (Control), a traditional Chinese medicine extract group (TCM) prepared in comparative example 1, a traditional Chinese medicine probiotic fermentation product group (TCMPM) prepared in example 2 and a monomeric compound group (TPMSC) with a structure of formula (I). The experimental group is pre-cultured for 4 days according to the dosage of 50 mug/mL, the phosphate buffer solution is used for replacing the traditional Chinese medicine probiotics fermentation metabolites in the normal control group, and counting is carried out for 1 time every day until all the worms die. The results are shown in fig. 12, the life of the N2 wild-type nematodes can be significantly prolonged by the traditional Chinese medicine probiotic fermentation product group and the monomer compound group with the structure of formula (i), and the life of the nematodes of the traditional Chinese medicine extract group, the traditional Chinese medicine probiotic fermentation product group and the monomer compound group with the structure of formula (i) can be respectively prolonged by 17.9%, 25.9% and 34.0% compared with the normal control group.

(2) The method comprises the following steps of culturing N2 wild-type nematodes at 20 ℃, randomly dividing the wild-type nematodes into 5 groups (the number of each group is not less than 60), and randomly dividing each group into 3 parallel Control groups, namely a normal Control group (abbreviated as Control), a model injury group (Par), a traditional Chinese medicine extract group (TCM) prepared in comparative example 1, a traditional Chinese medicine probiotic fermentation product group (TCMPM) prepared in example 2 and a monomeric compound group (TPMSC) with a structure of a formula (I), pre-culturing the experimental group for 4 days according to a dose of 50 mu g/mL, treating the nematodes in the other groups except the normal Control group by using paraquat, replacing the traditional Chinese medicines with phosphate buffer solutions in the normal Control group, counting 1 time every day for traditional Chinese medicine probiotic fermentation products and traditional Chinese medicine probiotic metabolic product monomeric compounds until all worms die. The results are shown in fig. 13, the life of the nematodes after paraquat treatment is significantly reduced, and the life of the nematodes after paraquat treatment is reduced by 23.9%; the service life of the nematodes cultured by the traditional Chinese medicine probiotic fermentation product and the monomeric compound with the structure of the formula (I) is obviously prolonged, and the service lives of the nematodes in the traditional Chinese medicine extract group, the traditional Chinese medicine probiotic fermentation product group and the monomeric compound with the structure of the formula (I) are respectively prolonged by 10.3%, 13.5% and 23.5%.

(3) Culturing N2 wild type nematodes at 20 deg.C, randomly dividing into 5 groups (each group has no less than 60), each group has 3 parallel Control groups, which are respectively a normal Control group (abbreviated as Control), a model injury group (Par), a traditional Chinese medicine extract group (TCM) prepared in comparative example 1, a traditional Chinese medicine probiotic fermentation product group (TCMPM) prepared in example 2 and a monomeric compound group (TPMSC) with formula (I), meanwhile, the experimental group was pre-cultured for 4 days at a dose of 50. mu.g/mL, except for the normal control group, treating other groups of nematodes with paraquat, replacing traditional Chinese medicine extract, traditional Chinese medicine probiotic fermentation product and monomer compound with formula (I) with phosphate buffered saline solution in normal control group, counting for 1 time every day, culturing for 4 days, and determining in vivo related antioxidant enzyme activity (superoxide dismutase SOD and malondialdehyde MDA) and Reactive Oxygen Species (ROS) content. After the traditional Chinese medicine extract, the traditional Chinese medicine probiotic fermentation product and the monomeric compound with the structure shown in the formula (I) are pre-cultured and treated by paraquat, the survival rate of the nematodes is recorded after the nematodes are continuously cultured for 4 days. The results are shown in FIGS. 14-16, wherein FIG. 14 is a graph of ROS content measurement, FIG. 15 is a graph of MDA content measurement, and FIG. 16 is a graph of SOD content measurement. After paraquat treatment, the activity of related antioxidant enzymes in the nematode bodies is obviously reduced compared with that of a normal control group, and the content of ROS is obviously increased compared with that of the normal control group; after being pretreated by the traditional Chinese medicine probiotic fermentation product of 50 mu g/mL and the monomer compound of the structure of the formula (I), the activity of the antioxidant enzyme in the nematode in the traditional Chinese medicine probiotic fermentation product group and the monomer compound of the structure of the formula (I) is obviously improved compared with that in a model damage group; for SOD activity, compared with a model damage group, the traditional Chinese medicine probiotic fermentation product group and the monomer compound group with the structure of the formula (I) are obviously increased; compared with a model damage group, the ROS content of the traditional Chinese medicine probiotic fermentation product group and the monomer compound group with the structure of the formula (I) is remarkably reduced, wherein in the monomer compound group with the structure of the formula (I), the activity of SOD is 1.26 times that of the model damage group, and the MDA content is 68.8% of that of the model damage group.

The experiments show that the anti-aging effect of the traditional Chinese medicine probiotic fermentation product and the monomeric compound with the structure of the formula (I) is greater than that of a simple traditional Chinese medicine extract; the traditional Chinese medicine probiotic fermentation product obtained by fermentation based on the traditional Chinese medicine extract and the monomer compound with the structure of the formula (I) have more excellent anti-aging effect; in particular to the monomer compound with the structure of formula (I) which is obtained by separation and has stronger anti-aging 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.

Claims

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:

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

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 to ferment for 24-48 h under the conditions that the temperature is 35-40 ℃ 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 0.1-0.3 as a mobile phase A, using a trifluoroacetic acid acetonitrile solution with the volume percent of 0.1-0.3 as a mobile phase B, wherein the mobile phase A: mobile phase B-78: 22; and (3) collecting the part corresponding to the chromatographic peak for 13.0-13.7 min, 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:

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 to ferment for 24-48 h under the conditions that the temperature is 35-40 ℃ 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 antioxidant and/or anti-aging effects;

the health care product has an anti-oxidation effect and/or an anti-aging effect.

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%.