Liver protection composition as well as application and product thereof

Abstract

The invention belongs to the technical field of nutritional compositions and biological medicines, and particularly relates to a liver protection composition as well as application and a product thereof. The liver protection composition provided by the invention consists of Akkermansia muciniphila, a silybum marianum extract and a globe artichoke extract, and the liver protection composition can be used for treating alcoholic, non-alcoholic fatty, chemical and autoimmune liver injury related diseases, such as alcoholic hepatitis, non-alcoholic fatty liver and the like, by virtue of the synergistic effect of the Akkermansia muciniphila, the silybum marianum extract and the globe artichoke extract. The traditional Chinese medicine composition is simple to prepare, can be prepared into functional food, health care products or medicines only by mixing according to a proportion, has both safety and effectiveness, and is suitable for liver injury treatment and daily liver health care.

Description

A liver-protective composition and its applications and products Technical Field

[0001] This invention belongs to the field of nutritional compositions and biomedical technology, specifically relating to a liver protection composition and its applications and products. Background Technology

[0002] As the metabolic center and vital detoxification organ of the human body, the liver undertakes key physiological functions such as substance transformation, toxin clearance, and immune regulation. Its health directly determines the stability of the body's overall physiological functions. However, with changes in modern lifestyles, increased exposure to environmental pollutants, and expanded clinical drug needs, liver damage has become a prevalent health problem worldwide. Among these, alcoholic liver injury, non-alcoholic fatty liver injury, and chemical liver injury are dominant, seriously threatening human health.

[0003] From the perspective of the causes of liver damage, excessive alcohol intake is the core factor leading to alcoholic liver injury. Long-term drinking causes ethanol to be metabolized in the liver to produce toxic substances such as acetaldehyde and reactive oxygen species, which trigger lipid peroxidation, inflammatory response, and mitochondrial dysfunction in hepatocytes, gradually developing into fatty liver, hepatitis, and even cirrhosis. Non-alcoholic fatty liver injury is closely related to high-sugar and high-fat diets, obesity, and metabolic syndrome. Excessive fat accumulation in hepatocytes will disrupt the balance of lipid metabolism in the liver, inducing endoplasmic reticulum stress and inflammatory cascade reactions. The causes of chemical liver injury are more extensive, including excessive use of clinical drugs such as acetaminophen, heavy metals in the environment (such as mercury and arsenic), pesticide residues, and industrial chemical pollutants (such as carbon tetrachloride). These substances can directly or indirectly damage the hepatocyte membrane structure, leading to hepatocyte necrosis and liver failure, and are more difficult to treat clinically.

[0004] To address the aforementioned liver damage issues, current liver protection methods mainly focus on three areas: chemotherapy, supplementation with single plant extracts, and probiotic regulation. However, all of these have significant limitations. In the field of chemotherapy, commonly used hepatoprotective drugs such as glycyrrhizic acid preparations and silymarin can inhibit hepatocyte inflammatory responses to some extent, but long-term use can easily lead to side effects such as sodium and water retention and elevated blood pressure, and their intervention effect on non-alcoholic fatty liver injury is limited. In the application of single plant extracts, due to the lack of synergistic effects between components, the bioavailability of their active ingredients is low, and they can only act on a specific pathological link in liver damage, making it difficult to achieve multi-target protection. The application of probiotics in liver protection is mostly limited to single strains (such as Lactobacillus and Bifidobacterium). These strains mainly improve the liver environment indirectly by regulating the balance of intestinal flora, but their ability to repair existing hepatocyte damage is weak, and they cannot cope with acute liver injury scenarios such as chemical liver injury.

[0005] Furthermore, current liver protection products generally suffer from issues such as single-ingredient composition, unclear mechanisms of action, and insufficient clinical validation. Most products only add a single plant extract or probiotic, failing to consider the synergistic effects between different active ingredients, leading to unstable liver-protective efficacy. Therefore, developing a liver protection composition with strong synergistic effects, comprehensive target targeting, high safety, and coverage of multiple types of liver injury has become an urgent need in the fields of nutritional composition and biomedicine, and is of great significance for improving liver protection efficacy and reducing the incidence of liver injury diseases. Summary of the Invention

[0006] To address the above shortcomings, the present invention provides a liver protection composition, its application, and products.

[0007] The technical solution of the present invention is as follows: On one hand, the present invention provides a liver protection composition, which is composed of Akkermansia muciniphila, milk thistle extract and artichoke extract.

[0008] Specifically, the liver protection composition comprises, by weight, 25-200 parts of Akkermansia myxophilus, 150-550 parts of milk thistle extract and 40-250 parts of artichoke extract.

[0009] More specifically, the liver-protective composition comprises, by weight, 25-30 parts, 30-35 parts, 25-30 parts, 30-35 parts, 35-40 parts, 40-45 parts, 45-50 parts, 50-55 parts, 55-60 parts, 60-65 parts, 65-70 parts, 70-75 parts, 75-80 parts, 80-85 parts, 85-90 parts, 90-95 parts, 95-100 parts, 100-105 parts, 105-110 parts, 110- 115, 115-120, 120-125, 125-130, 130-135, 135-140, 140-145, 145-150, 150-155, 155-160, 160-165, 165-170, 170-175, 175-180, 180-185, 185-190, 190-195, or 195-200 samples of Akkermansia myxophilus.

[0010] Preferably, the liver-protective composition comprises, by weight, 25-30 parts, 30-35 parts, 25-30 parts, 30-35 parts, 35-40 parts, 40-45 parts, 45-50 parts, 50-55 parts, 55-60 parts, 60-65 parts, 65-70 parts, 70-75 parts, 75-80 parts, 80-85 parts, 85-90 parts, 90-95 parts, 95-100 parts, or 100-105 parts. More preferably, the liver protection composition contains 40-45, 45-50, 50-55, or 55-60 parts of Akkermansia myxophilus by weight, in parts of weight.

[0011] More specifically, the liver-protective composition comprises, by weight, 150-155 parts, 155-160 parts, 160-165 parts, 165-170 parts, 170-175 parts, 175-180 parts, 180-185 parts, 185-190 parts, 190-195 parts, 195-200 parts, 200-205 parts, 205-210 parts, 210-215 parts, 215-220 parts, 220-225 parts, 225-230 parts, 230-235 parts, 235-240 parts, 240-245 parts, 245-250 parts, 250-255 parts, 255- 260 portions, 260-265 portions, 265-270 portions, 270-275 portions, 275-280 portions, 280-285 portions, 285-290 portions, 290-295 portions, 295-300 portions, 300-305 portions, 305-310 portions, 310-315 portions, 315-320 portions, 320-325 portions, 325-330 portions, 330-335 portions, 335-340 portions, 340-345 portions, 345-350 portions, 350-355 portions, 35 5-360 portions, 360-365 portions, 365-370 portions, 370-375 portions, 375-380 portions, 380-385 portions, 385-390 portions, 390-395 portions, 395-400 portions, 400-405 portions, 405-410 portions, 410-415 portions, 415-420 portions, 420-425 portions, 425-430 portions, 430-435 portions, 435-440 portions, 440-445 portions, 445-450 portions, 450-455 portions 455-460 parts, 460-465 parts, 465-470 parts, 470-475 parts, 475-480 parts, 480-485 parts, 485-490 parts, 490-495 parts, 495-500 parts, 500-505 parts, 505-510 parts, 510-515 parts, 515-520 parts, 520-525 parts, 525-530 parts, 530-535 parts, 535-540 parts, 540-545 parts, or 545-550 parts milk thistle extract.

[0012] Preferably, the liver-protective composition comprises, by weight, 200-205 parts, 205-210 parts, 210-215 parts, 215-220 parts, 220-225 parts, 225-230 parts, 230-235 parts, 235-240 parts, 240-245 parts, 245-250 parts, 250-255 parts, 255-260 parts, 260-265 parts, 265-270 parts, 270-270 parts, etc. 5 servings, 275-280 servings, 280-285 servings, 285-290 servings, 290-295 servings, 295-300 servings, 300-305 servings, 305-310 servings, 310-315 servings, 315-320 servings, 320-325 servings, 325-330 servings, 330-335 servings, 335-340 servings, 340-345 servings, 345-350 servings, 350-355 servings, 355-36 servings 0 portions, 360-365 portions, 365-370 portions, 370-375 portions, 375-380 portions, 380-385 portions, 385-390 portions, 390-395 portions, 395-400 portions, 400-405 portions, 405-410 portions, 410-415 portions, 415-420 portions, 420-425 portions, 425-430 portions, 430-435 portions, 435-440 portions, 440-445 portions Milk thistle extract in 5 parts, 445-450 parts, 450-455 parts, 455-460 parts, 460-465 parts, 465-470 parts, 470-475 parts, 475-480 parts, 480-485 parts, 485-490 parts, 490-495 parts, 495-500 parts, 500-505 parts, 505-510 parts, 510-515 parts, 515-520 parts, or 520-525 parts.

[0013] More preferably, the liver-protective composition comprises 500-505 parts, 505-510 parts, 510-515 parts, 515-520 parts, or 520-525 parts of milk thistle extract by weight.

[0014] More specifically, the liver-protective composition comprises, by weight, 40-45 parts, 45-50 parts, 50-55 parts, 55-60 parts, 60-65 parts, 65-70 parts, 70-75 parts, 75-80 parts, 80-85 parts, 85-90 parts, 90-95 parts, 95-100 parts, 100-105 parts, 105-110 parts, 110-115 parts, 115-120 parts, 120-125 parts, 125-130 parts, 130-135 parts, 135-140 parts, 140-145 parts, 14 5-150 parts, 150-155 parts, 155-160 parts, 160-165 parts, 165-170 parts, 170-175 parts, 175-180 parts, 180-185 parts, 185-190 parts, 190-195 parts, 195-200 parts, 200-205 parts, 205-210 parts, 210-215 parts, 215-220 parts, 220-225 parts, 225-230 parts, 230-235 parts, 235-240 parts, 240-245 parts, or 245-250 parts of artichoke extract.

[0015] Preferably, the liver-protective composition comprises, by weight, 40-45 parts, 45-50 parts, 50-55 parts, 55-60 parts, 60-65 parts, 65-70 parts, 70-75 parts, 75-80 parts, 80-85 parts, 85-90 parts, 90-95 parts, 95-100 parts, 100-105 parts, 105-110 parts, 110-115 parts, 115-120 parts, or 120-125 parts of artichoke extract.

[0016] More preferably, the liver-protective composition comprises 40-45 parts, 45-50 parts, 50-55 parts, or 55-60 parts of artichoke extract by weight.

[0017] Specifically, the *Akkermansia* species mentioned include, but are not limited to: AKK PROBIO and *Akkermansia MucT*. TM Any one or more of Akk 11.

[0018] Preferably, the *Akkermansia* species is selected from: AKK PROBIO and *Akkermansia mucT*. TM Any one or more of Akk11.

[0019] The AKK PROBIO mentioned has the accession number CGMCC No.20955.

[0020] The Akkermansia MucT TM The accession number is: ATCC BAA-835.

[0021] The accession number of the AKK 11 is: CCTCC: M 2024119.

[0022] Specifically, the Akkermansia myxophilus includes: live Akkermansia myxophilus and / or inactivated Akkermansia myxophilus.

[0023] Preferably, the concentration of viable Akkermansia myxophilus is 1 × 10⁻⁶. 11 -1×10 12 CFU / g.

[0024] More preferably, the concentration of viable Akkermansia myxophilus is 2 × 10⁻⁶. 11 CFU / g.

[0025] Preferably, the concentration of inactivated Akkermansia myxophilus is 1 × 10⁻⁶. 11 -1×10 12 TFU / g.

[0026] More preferably, the concentration of inactivated Akkermansia myxophilus is 2 × 10⁻⁶. 11 TFU / g.

[0027] On the other hand, the present invention provides a method for preparing the liver protection composition described in any of the above claims.

[0028] Specifically, the preparation method includes mixing Akkermansia muciniphila, milk thistle extract, and artichoke extract in parts by weight.

[0029] Specifically, the preparation method of milk thistle extract includes: (1) milk thistle seeds are put into an oil press to remove the oil in the seeds and obtain milk thistle cake; (2) an extraction solvent is added to the milk thistle cake and extracted to obtain an extract; (3) the extract is concentrated to obtain a concentrate; (4) the concentrate is defatted to obtain milk thistle extract; (5) the milk thistle extract is dried after removing the residual volatile components in the material to obtain milk thistle extract.

[0030] Preferably, the mass ratio of milk thistle cake meal to extraction solvent in step (2) is 1:1-15.

[0031] More preferably, the mass ratio of milk thistle cake to extraction solvent in step (2) is 1:1.5.

[0032] Preferably, the extraction solvent in step (2) is any one or more of ethyl acetate, ethanol, and acetone.

[0033] More preferably, the extraction solvent in step (2) is ethyl acetate.

[0034] More preferably, the ethyl acetate is ethyl acetate of 90% v / v to 95% v / v.

[0035] Preferably, the extraction in step (2) is performed at 60-80℃ for 10-30 hours.

[0036] Preferably, the concentration in step (3) is maintained at 75-100°C for 15-20 hours.

[0037] More preferably, the concentration in step (3) is maintained at 75-100°C for 16 hours.

[0038] More preferably, the concentration in step (3) is maintained at 75-85℃ for 13.5h, and the temperature is adjusted to 90-100℃ and maintained for 2.5h.

[0039] Preferably, the degreasing in step (4) is as follows: after the concentrate has been allowed to stand, the precipitated oil is removed; a degreasing solvent is added, stirred, and allowed to stand, then the degreasing solvent is removed. The above degreasing steps are repeated twice to obtain milk thistle extract.

[0040] More preferably, the amount of the degreasing solvent added is 1-2 times the volume of the concentrated liquid.

[0041] More preferably, the degreasing solvent includes any one or more of acetone, ethyl acetate, and ethanol.

[0042] More preferably, the degreasing solvent is acetone.

[0043] More preferably, the settling period is at least 30 minutes.

[0044] More preferably, the stirring and then standing refers to stirring for at least 10 minutes and then standing for at least 10 minutes.

[0045] Preferably, the method for removing residual volatile components from the material in step (5) includes the following steps: 1) Temperature 50-70℃, vacuum degree -0.01 to -0.03 MPa, maintain for 20-40 min; 2) Stirring, temperature rise to 60-80℃, maintain for 20-40 min; 3) After the foam disappears, temperature rise to 80-95℃, vacuum degree controlled at -0.03 to -0.06 MPa, maintain for 100-150 min.

[0046] More preferably, step 1) is a temperature of 60-70℃, a vacuum of -0.01 to -0.03MPa, and is maintained for 20-40 minutes.

[0047] More preferably, step 1) is a temperature of 60-70℃, a vacuum degree of -0.01 to -0.03MPa, and a holding time of 30min.

[0048] More preferably, step 2) involves stirring, with the temperature raised to 70-80°C and maintained for 20-40 minutes; even more preferably, step 2) involves stirring, with the temperature raised to 70-80°C and maintained for 30 minutes.

[0049] More preferably, step 3) involves raising the temperature to 85-95°C after the foam disappears, controlling the vacuum level at -0.03 ~ -0.06 MPa, and maintaining this temperature for 100-150 minutes.

[0050] More preferably, step 3) involves raising the temperature to 85-95℃ after the foam disappears, controlling the vacuum degree at -0.03~-0.06Mpa, and maintaining this temperature for 120min.

[0051] Preferably, the drying in step (5) is vacuum drying.

[0052] Specifically, the preparation method of the artichoke extract includes: S1, extracting artichoke leaves twice with water and combining the extracts; S2, filtering and concentrating the extract to obtain a concentrated solution; S3, freeze-drying the concentrated solution, pulverizing it, and sieving it to obtain the artichoke extract.

[0053] Preferably, the amount of water added in step S1 is 5-15 times the weight of the artichoke leaves.

[0054] More preferably, the amount of water added in step S1 is 10 times the weight of the artichoke leaves.

[0055] Preferably, the extraction conditions in step S1 are extraction at 80-90℃ for 1.5 hours.

[0056] Preferably, the concentration in step S2 is to concentrate to a relative density of 1.1-1.3 at 50°C.

[0057] In another aspect, the present invention provides a functional food or health product comprising the liver-protecting composition described in any one of the above claims.

[0058] Specifically, the dosage forms of the functional foods or health products include liquid dosage forms, solid dosage forms, or semi-solid dosage forms.

[0059] Preferably, the food products include, but are not limited to: candy, soy milk, yogurt, canned food, biscuits, chocolate, pastries, cream, cheese, dairy products, milk powder, formula milk powder, ice cream, jam, fruit puree, candied fruit, preserved fruit, dried fruit, bread, egg rolls, protein drinks, solid beverages, lactic acid bacteria drinks, plant protein drinks, carbonated drinks, coffee, or puffed foods.

[0060] Preferably, the dosage form of the health product includes, but is not limited to: tablets, capsules, soft capsules, granules, pills, gel candies, powders, oral liquids, or drops.

[0061] Specifically, the functional foods or health products mentioned also include nutritionally acceptable nutrients.

[0062] Preferably, the nutrients include, but are not limited to, any one or more of the following: dietary fiber, prebiotics, protein, lipids, minerals, and vitamins.

[0063] In another aspect, the present invention provides a medicine comprising the liver-protecting composition described in any of the preceding claims.

[0064] Specifically, the dosage form of the medicine includes any one or more of the following: tablets, pills, powders, suspensions, gels, emulsions, creams, granules, capsules, suppositories, injections, sprays, and injections.

[0065] Specifically, the medicine also includes pharmaceutically acceptable excipients.

[0066] Preferably, the pharmaceutically acceptable excipients include one or more of the following: wetting agents, emulsifiers, preservatives, antioxidants, buffers, diluents, lubricants, solutes, suspending agents, solubilizers, thickeners, stabilizers, sweeteners, and flavorings.

[0067] In another aspect, the present invention provides the use of the liver protection composition described in any one of the above claims, the use including any one or more of the following: (1) use in the preparation of functional foods or health products that have an auxiliary protective effect against chemically induced liver injury; (2) use in the preparation of medicaments for the prevention or treatment of liver injury.

[0068] Preferably, the drugs for preventing or treating liver injury include any one or more of the following: drugs for preventing or treating alcoholic liver injury, drugs for preventing or treating chemically induced liver injury, drugs for preventing or treating non-alcoholic fatty liver injury, and drugs for preventing or treating autoimmune liver injury.

[0069] In another aspect, the present invention provides a method for preventing or treating liver injury, the method comprising using any of the liver-protecting compositions, foods, health products or pharmaceuticals described above.

[0070] Specifically, the method includes administering an effective amount of a liver-protective composition, food, health product, or medicine to the subject.

[0071] Preferably, the subject is a mammal.

[0072] More preferably, the mammal is a human.

[0073] The beneficial effects of this invention are as follows: The liver-protective composition provided by this invention consists of Akkermansia myxophilus, milk thistle extract, and artichoke extract. This liver-protective composition, relying on the synergistic effect of these three ingredients, can treat alcoholic, non-alcoholic fatty, chemical, and autoimmune liver injury-related diseases, such as alcoholic hepatitis and non-alcoholic fatty liver disease. Its preparation is simple, requiring only proportional mixing, and it can be formulated into functional foods, health products, or pharmaceuticals, balancing safety and efficacy, and is suitable for liver injury treatment and daily liver health maintenance. Attached Figure Description

[0074] Figure 1 shows the adjunctive protective effect against alcoholic fatty liver disease: phenotypic evaluation.

[0075] Figure 2 shows the auxiliary protective effect against alcoholic fatty liver: evaluation of liver pathological sections.

[0076] Figure 3 shows the adjunctive protective effect against food-induced fatty liver: phenotypic evaluation.

[0077] Figure 4 shows the auxiliary protective effect against food-induced neutrophil damage.

[0078] Figure 5 shows the auxiliary protective effect induced by food: evaluation of liver pathological sections. Detailed Implementation

[0079] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0080] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0081] Basic Example 1: Preparation of *Ackermania myxophilus* The strain used in this invention is *Ackermania myxophilus*. Live bacteria: *Ackermania myxophilus* seed culture at a ratio of 10... 6 CFU / mL, inoculated into thioglycolate liquid medium, anaerobically cultured at 37℃ for 15 h, centrifuged at 4000 rpm to collect bacterial sludge, freeze-dried, pulverized and sieved for later use, with a bacterial concentration of 2×10⁻⁶. 11 CFU / g yielded live bacterial powder. Inactivated bacteria: Akkermansia myxophilus seed culture at 10... 6CFU / mL, inoculated into thioglycolate liquid medium, anaerobic cultured at 37℃ for 15 h, pasteurized at 65℃ for 30 min, centrifuged at 4000 rpm to collect bacterial sludge, freeze-dried, pulverized and sieved for later use, with a bacterial concentration of 2×10⁻⁶. 11 TFU / g yields inactivated bacterial powder.

[0082] The Akkermansia muciniphila strain involved in this invention covers all strains taxonomically belonging to Akkermansia muciniphila; in the examples, AKK PROBIO (CGMCC No. 20955, Shanenkang Biotechnology (Suzhou) Co., Ltd., batch number W2409088) and Akkermansia MucT... TM (ATCC BAA-835, The Akkermansia Company SA, batch number E024776A) and Akk 11 (CCTCC: M 2024119, Microcare Probiotics (Suzhou) Co., Ltd., batch number: YF202504181001) are described as representative strains, but they are not intended to limit the specific species of Akkermansia myxophilus protected by this invention.

[0083] Basic Example 2 Preparation of Milk Thistle Extract 1. Milk thistle seeds are placed in an oil press to remove the oil from the seeds, resulting in milk thistle cake.

[0084] 2. Add 95% v / v ethyl acetate to the milk thistle cake at a mass ratio of 1:1.5 (milk thistle cake to extraction solvent), and reflux at 60℃ for 20 hours to obtain the extract. 3. Place the extract in a concentration tank and maintain at 85℃ for 13.5 hours, then adjust the temperature to 100℃ and maintain for 2.5 hours to obtain the concentrate. 4. Degreasing: Transfer the concentrate to a degreasing and residue removal machine, let it stand for 30 minutes, and remove the precipitated oil. Add acetone at twice the volume of the concentrate, stir for 10 minutes, let it stand for 10 minutes, and remove the acetone. Repeat the above degreasing steps twice more to obtain milk thistle extract.

[0085] 5. Heat the milk thistle extract, maintaining the tank temperature at 60℃ and the vacuum at -0.01 to -0.03 MPa for 30 minutes. Start stirring and raise the tank temperature to 70℃, maintaining this temperature for 30 minutes. Once the foam disappears, maintain the vacuum between -0.03 and -0.06 MPa and the temperature at 85℃ for 120 minutes to complete the residue removal process.

[0086] 6. The extract after removing residues is vacuum dried to obtain milk thistle extract.

[0087] The milk thistle extract claimed in this invention is not limited to the preparation method. The preparation method described in this basic embodiment is for illustrative purposes only. Milk thistle extracts prepared by reasonable adjustments or improvements made by those skilled in the art based on this exemplary method should all fall within the protection scope of this invention.

[0088] Basic Example 3: Preparation of Artichoke Extract 1. Artichoke leaves were extracted with 10 times their weight of water at an extraction temperature of 80-90℃, refluxed twice for 1.5 hours each time to obtain an extract; 2. The extract was filtered and the filtrates were combined; 3. The filtered extract was concentrated to a relative density of 1.1-1.3 at 50℃ to obtain a concentrate; 4. The concentrate was freeze-dried for 48 hours, then pulverized and sieved to obtain the artichoke extract.

[0089] The artichoke extract claimed in this invention is not limited to the preparation method. The preparation method described in this basic embodiment is for illustrative purposes only. Artichoke extracts prepared by reasonable adjustments or improvements made by those skilled in the artichoke based on this exemplary method should all fall within the protection scope of this invention.

[0090] Example 1: A liver protection composition, wherein the liver protection composition comprises, by weight, 50 parts of AKK PROBIO live bacteria powder, 525 parts of milk thistle extract and 40 parts of artichoke extract.

[0091] The liver protection composition is prepared by mixing AKK PROBIO live bacteria powder, milk thistle extract and artichoke extract in proportion to weight to obtain the liver protection composition.

[0092] Example 2: A liver-protective composition, wherein the liver-protective composition comprises 50 parts by weight of Akkermansia MucT TM It consists of live bacteria powder, 525 parts milk thistle extract and 40 parts artichoke extract.

[0093] The method for preparing the liver-protective composition is as follows: Akkermansia MucT TM Live bacteria powder, milk thistle extract and artichoke extract were mixed evenly in parts by weight to obtain a liver-protective composition.

[0094] Example 3: A liver protection composition, wherein the liver protection composition comprises, by weight, 50 parts Akk 11 live bacteria powder, 525 parts milk thistle extract and 40 parts artichoke extract.

[0095] The liver protection composition is prepared by mixing Akk 11 live bacteria powder, milk thistle extract and artichoke extract in proportion to weight to obtain the liver protection composition.

[0096] Example 4: A liver protection composition, wherein the liver protection composition comprises, by weight, 25 parts AKK PROBIO inactivated bacterial powder, 550 parts milk thistle extract and 40 parts artichoke extract.

[0097] The preparation method of the liver-protecting composition is as described in Example 1.

[0098] Example 5: A liver protection composition, wherein the liver protection composition comprises, by weight, 200 parts AKK PROBIO live bacteria powder, 150 parts milk thistle extract and 250 parts artichoke extract.

[0099] The preparation method of the liver-protecting composition is as described in Example 1.

[0100] Comparative Example 1: A liver-protecting composition comprising, by weight, 525 parts milk thistle extract and 90 parts artichoke extract.

[0101] The liver-protecting composition is prepared by mixing milk thistle extract and artichoke extract in proportion to weight to obtain the liver-protecting composition.

[0102] Comparative Example 2: A liver protection composition, wherein the liver protection composition comprises, by weight, 50 parts of AKK PROBIO live bacteria powder and 40 parts of artichoke extract.

[0103] The liver protection composition is prepared by mixing AKK PROBIO live bacteria powder and artichoke extract in proportion to weight to obtain the liver protection composition.

[0104] Comparative Example 3: A liver protection composition comprising, by weight, 50 parts of AKK PROBIO live bacteria powder, 525 parts of milk thistle extract and 40 parts of puerarin.

[0105] The liver protection composition is prepared by mixing AKK PROBIO live bacteria powder, milk thistle extract and puerarin in proportion to weight to obtain the liver protection composition.

[0106] Comparative Example 41: Milk thistle seeds were placed in an oil press to remove the oil from the seeds, resulting in milk thistle cake.

[0107] 2. Add 95% v / v ethyl acetate to the milk thistle cake at a mass ratio of 1:1.5 (milk thistle cake to extraction solvent), and reflux at 60℃ for 20 hours to obtain the extract. 3. Place the extract in a concentration tank and maintain at 85℃ for 13.5 hours, then adjust the temperature to 100℃ and maintain for 2.5 hours to obtain the concentrate. 4. Degreasing: Transfer the concentrate to a degreasing and residue removal machine, let it stand for 30 minutes, and remove the precipitated oil. Add acetone at twice the volume of the concentrate, stir for 10 minutes, let it stand for 10 minutes, and remove the acetone. Repeat the above degreasing steps twice more to obtain milk thistle extract.

[0108] 5. Heat the milk thistle extract, maintaining the tank temperature at 60℃ and the vacuum at -0.01 to -0.03 MPa for 30 minutes. Start stirring and raise the tank temperature to 70℃, maintaining this temperature for 30 minutes. Once the foam disappears, maintain the vacuum between -0.03 and -0.06 MPa and the temperature at 85℃ for 120 minutes to complete the residue removal process.

[0109] 6. The extract after removing residues is vacuum dried to obtain milk thistle extract.

[0110] Comparative Example 51: 1. Artichoke leaves were extracted with 10 times their weight of water at an extraction temperature of 80-90℃, refluxed twice for 1.5 hours each time to obtain an extract; 2. The extract was filtered and the filtrates were combined; 3. The filtered extract was concentrated to a relative density of 1.1-1.3 at 50℃ to obtain a concentrate; 4. The concentrate was freeze-dried for 48 hours, then pulverized and sieved to obtain the artichoke extract.

[0111] Experimental Example 1: Adjunctive Protective Efficacy Against Alcoholic Fatty Liver 1. Test Samples: The test samples were the compositions of Examples 1-5 or Comparative Examples 1-3, milk thistle extract of Comparative Example 4, artichoke extract of Comparative Example 5, and curcumin (positive control). The experimental concentrations of the test samples are shown in Table 1.

[0112] Table 1

[0113] 2. All experimental zebrafish were raised in aquarium water at 28℃ (water quality: 200mg of instant sea salt added per 1L of reverse osmosis water, conductivity 450~550μS / cm; pH 6.5-8.5; hardness 50-100mg / L CaCO3), bred and provided by Huante Biological Fish Farming Center. The experimental animal use license number is: SYXK (Zhejiang) 2022-0004. The husbandry and management met the requirements of international AAALAC certification (certification number: 001458), and the IACUC ethics review number is: IACUC-2025-9601-01.

[0114] 3. Evaluation of the adjunctive protective efficacy against alcoholic fatty liver (phenotype): 5-dpf melanin allele mutant zebrafish (albino) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Samples were administered via water-soluble solution, along with a positive control (curcumin). A normal control group and a model control group were also included, with a volume of 3 mL per well. Except for the normal control group, all other experimental groups were treated with anhydrous ethanol to establish a zebrafish alcoholic fatty liver model. After treatment at 28℃ for 1 day, samples were collected and stained with Oil Red O. Ten zebrafish from each experimental group were randomly selected and photographed under a dissecting microscope. Data were collected using NIS-ElementsD 3.20 advanced image processing software, and the intensity of liver fat staining was analyzed. The statistical analysis results of this index were used to evaluate the adjunctive protective efficacy of the samples against alcoholic fatty liver. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS software; p < 0.05 indicated statistical significance.

[0115] The measurement results are shown in Figure 1 and Table 2: Table 2

[0116] #p<0.05, compared with the control group; *p<0.05, compared with the model group; &p<0.05, compared with comparative examples 1-5.

[0117] 4. Evaluation of the adjunctive protective efficacy against alcoholic fatty liver (ALDH, ADH): Wild-type AB strain zebrafish, 5 days post-fertilization (5 dpf), were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Samples were administered in water, along with a positive control (curcumin). A normal control group and a model control group were also included, with a volume of 3 mL per well. The experiment was conducted in triplicate. Except for the normal control group, all experimental groups were treated with anhydrous ethanol to establish a zebrafish alcoholic fatty liver model. After treatment at 28℃ for 1 day, ALDH and ADH assay kits were used, and data were collected using a multi-functional microplate reader. The activities of ALDH and ADH in zebrafish were analyzed, and the statistical analysis results of these indicators were used to evaluate the adjunctive protective efficacy against alcoholic fatty liver. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS software; p < 0.05 indicated statistical significance.

[0118] The measurement results are shown in Table 3: Table 3

[0119] #p<0.05, compared with the control group; *p<0.05, compared with the model group; &p<0.05, compared with comparative examples 1-5.

[0120] 5. Evaluation of the adjunctive protective efficacy against alcoholic fatty liver (liver pathological sections): Wild-type AB strain zebrafish (5 dpf) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Samples were administered in water, with curcumin as a positive control. A normal control group and a model control group were also included, with a volume of 3 mL per well. Except for the normal control group, all other experimental groups were treated with anhydrous ethanol to establish a zebrafish alcoholic fatty liver model. After treatment at 28℃ for 1 day, the zebrafish were fixed with 4% histocellular fixative. Following a series of steps including dehydration, embedding, sectioning, and staining, the zebrafish underwent histopathological H&E staining analysis. The adjunctive protective efficacy of the samples against alcoholic fatty liver was evaluated through liver histopathological analysis. The results are shown in Figure 2.

[0121] Experimental Example 2: Adjunctive Protective Efficacy Against Food-Induced Fatty Liver 1. Test Samples: The test samples were the compositions of Examples 1-5 or Comparative Examples 1-3, milk thistle extract of Comparative Example 4, artichoke extract of Comparative Example 5, and positive control atorvastatin calcium. The experimental concentrations of the test samples are shown in Table 4.

[0122] Table 4

[0123] 2. All experimental zebrafish were raised in aquarium water at 28℃ (water quality: 200mg of instant sea salt added per 1L of reverse osmosis water, conductivity 450-550μS / cm; pH 6.5-8.5; hardness 50-100mg / L CaCO3), bred and provided by Huante Biological Fish Farming Center. The experimental animal use license number is: SYXK (Zhejiang) 2022-0004. The husbandry and management met the requirements of international AAALAC certification (certification number: 001458), and the IACUC ethics review number is: IACUC-2025-9601-01.

[0124] 3. Evaluation of the auxiliary protective efficacy against food-induced fatty liver (phenotype): 5-day-fed zebrafish (albino) with a melanin allele mutation were randomly selected and placed in beakers, with 30 zebrafish treated in each beaker (experimental group). Samples were administered via water-soluble solution, along with atorvastatin calcium as a positive control. A normal control group and a model control group were also included. Each beaker contained 25 mL. Except for the normal control group, all other experimental groups were administered a high-sugar, high-fat diet via water-soluble solution to establish a zebrafish food-induced fatty liver model. After treatment at 28℃ for 2 days, samples were collected and stained with Oil Red O. Ten zebrafish from each experimental group were randomly selected and photographed under a dissecting microscope. Data were collected using NIS-Elements D3.20 advanced image processing software, and the intensity of liver fat staining was analyzed. The statistical analysis results of this index were used to evaluate the auxiliary protective efficacy of the samples against food-induced fatty liver. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS software; p < 0.05 indicated statistical significance.

[0125] The measurement results are shown in Figure 3 and Table 5: Table 5

[0126] #p<0.05, compared with the control group; *p<0.05, compared with the model group; &p<0.05, compared with comparative examples 1-5.

[0127] 4. Evaluation of the auxiliary protective efficacy against food-induced fatty liver (neutrophils): 5-dpf transgenic green fluorescent zebrafish (MPX) with neutrophils were randomly selected and placed in beakers, with 30 zebrafish treated in each beaker (experimental group). Samples were administered in water, along with a positive control (atorvastatin calcium). A normal control group and a model control group were also included. Each beaker had a volume of 25 mL. Except for the normal control group, all experimental groups were given a high-sugar, high-fat diet to establish a zebrafish food-induced fatty liver model. After treatment at 28℃ for 2 days, 10 zebrafish from each experimental group were randomly selected and photographed under a fluorescence microscope. Data were collected using NIS-ElementsD3.20 advanced image processing software, and the number of neutrophils in the zebrafish liver was analyzed. The statistical analysis results of this index were used to evaluate the auxiliary protective efficacy of the samples against food-induced fatty liver. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS software; p < 0.05 indicated statistical significance.

[0128] The measurement results are shown in Figure 4 and Table 6: Table 6

[0129] #p<0.05, compared with the control group; *p<0.05, compared with the model group; &p<0.05, compared with comparative examples 1-5.

[0130] 5. Evaluation of the auxiliary protective efficacy against food-induced fatty liver (AST, ALT): Wild-type AB strain zebrafish (5 dpf) were randomly selected and placed in beakers, with 30 zebrafish treated in each beaker (experimental group). Samples were administered via water-soluble solution, along with atorvastatin calcium as a positive control. A normal control group and a model control group were also included. Each beaker contained 25 mL. The experiment was conducted in triplicate. Except for the normal control group, all experimental groups were administered a high-sugar, high-fat diet via water-soluble solution to establish a zebrafish food-induced fatty liver model. After treatment at 28℃ for 2 days, ALT and AST assay kits were used, and data were collected using a multi-functional microplate reader to analyze the ALT and AST activities in the zebrafish. The statistical analysis results of these indicators were used to evaluate the auxiliary protective efficacy of the samples against food-induced fatty liver. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS software; p < 0.05 indicated statistical significance.

[0131] The measurement results are shown in Table 7: Table 7

[0132] #p<0.05, compared with the control group; *p<0.05, compared with the model group; &p<0.05, compared with comparative examples 1-5.

[0133] 6. Evaluation of the auxiliary protective efficacy against food-induced fatty liver (liver histopathological sections): Wild-type AB strain zebrafish (5 dpf) were randomly selected and placed in beakers, with 30 zebrafish treated in each beaker (experimental group). Samples were administered in water, along with atorvastatin calcium as a positive control. A normal control group and a model control group were also included. Each beaker contained 25 mL. Except for the normal control group, all experimental groups were given a high-sugar, high-fat diet to establish a zebrafish food-induced fatty liver model. After treatment at 28℃ for 2 days, the zebrafish were fixed with 4% histopathological fixative. Following a series of steps including dehydration, embedding, sectioning, and staining, the zebrafish underwent histopathological H&E staining analysis. The auxiliary protective efficacy of the samples against food-induced fatty liver was evaluated through liver histopathological analysis. The results are shown in Figure 5.

[0134] Experiment Example 3: Adjunctive Protective Effect on Chemically Induced Liver Injury 1. Experimental Grouping: Eight-week-old male C57BL / 6J mice were selected and placed in an acclimatization environment for one week. During the rearing period, they were provided with normal food and free access to water. The rearing conditions were controlled at a temperature of (22 ± 2)℃, humidity of (50 ± 10)%, and a 12-hour light-dark cycle. They were randomly divided into 13 groups, including a blank control group, a model group, a positive control group, Example 1-Example 5 groups, and Comparative Example 1-Comparative Example 5 groups, with 10 mice in each group.

[0135] 2. Experimental Procedure: Blank group: mice were administered an equal volume of PBS by gavage; Model group: mice were administered an equal volume of PBS by gavage; Positive control group: mice were administered 300 mg / kg acetylcysteine ​​by gavage; Example 1 group: mice were administered 10 mg / kg of the liver protection composition of Example 1 by gavage.

[0136] Example 2 group: Mice were administered the liver-protective composition of Example 2 by gavage at a dose of 10 mg / kg.

[0137] Example 3 group: Mice were administered the liver-protective composition of Example 3 by gavage at a dose of 10 mg / kg.

[0138] Comparative Example 1: Mice were administered 10 mg / kg of the liver-protective composition of Comparative Example 1 by gavage.

[0139] Comparative Example 2: Mice were administered 10 mg / kg of the liver-protective composition of Comparative Example 2 by gavage.

[0140] Comparative Example 3: Mice were administered 10 mg / kg of the liver-protective composition of Comparative Example 3 by gavage.

[0141] Comparative Example 4: Mice were administered 10 mg / kg of milk thistle extract (Comparative Example 4) by gavage; Comparative Example 5: Mice were administered 10 mg / kg of artichoke extract (Comparative Example 5) by gavage.

[0142] Mice were administered the drugs via gavage according to the above-mentioned groups and dosages, once daily for 7 consecutive days. Mice were weighed after the last gavage. One hour after the last gavage, an acute liver injury model was established in mice (mice were fasted for 12 hours prior to modeling to reduce interference from the contents). Mice were intraperitoneally injected with 300 mg / kg of acetaminophen to establish the acute liver injury model; the control group received the same volume of PBS intraperitoneally. Twenty-four hours after the intraperitoneal injection of acetaminophen, blood was collected from the mice by enucleation using enzyme-free centrifuge tubes for AST and ALT determination. Mice were euthanized by cervical dislocation, and the livers were collected and weighed. The collected livers were stored at -80°C for subsequent detection of inflammatory factor levels.

[0143] 3. Serum ALT and AST Measurement (n=10): Mouse blood was centrifuged at 5000 rpm at 4°C for 10 min, and the supernatant was collected to obtain mouse serum samples. Serum ALT and AST levels were measured using AST and ALT kits. Mouse hepatic lobes were dehydrated and embedded for subsequent H&E staining and other pathological examinations. The remaining liver fragments were cut into small pieces and stored at -80°C. Oxidative stress and inflammation-related markers were detected using qPCR and Western blotting.

[0144] The results showed that Examples 1-3 significantly reduced the levels of ALT and AST in serum, with better results than Comparative Examples 1-5.

[0145] The above detailed description is a specific illustration of one feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or modifications made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. A liver-protective composition, characterized in that, The liver-protective composition comprises Akkermansia muciniphila, milk thistle extract, and artichoke extract.

2. The liver-protective composition according to claim 1, characterized in that, The liver protection composition comprises, by weight, 25-200 parts of Akkermansia myxophilus, 150-550 parts of milk thistle extract and 40-250 parts of artichoke extract.

3. The liver-protective composition according to claim 1, characterized in that, The liver protection composition comprises, by weight, 50-150 parts of Akkermansia myxophilus, 200-525 parts of milk thistle extract and 40-125 parts of artichoke extract.

4. The composition according to claim 1, characterized in that, The aforementioned Akkermansia species include AKKPROBIO and Akkermansia MucT. TM Any one or more of Akk 11.

5. The liver-protective composition according to claim 1, characterized in that, The aforementioned Akkermansia myxophilus includes: live Akkermansia myxophilus and / or inactivated Akkermansia myxophilus.

6. The liver-protective composition according to claim 5, characterized in that, The concentration of viable Akkermansia myxophilus was 1 × 10⁻⁶. 11 -1×10 12 CFU / g, the concentration of inactivated Akkermansia myxophilus is 1×10⁻⁶. 11 -1×10 12 TFU / g.

7. The liver-protective composition according to claim 6, characterized in that, The concentration of viable Akkermansia myxophilus was 2 × 10⁻⁶. 11 CFU / g, the concentration of inactivated Akkermansia myxophilus was 2×10⁻⁶. 11 TFU / g.

8. A method for preparing the liver-protective composition according to any one of claims 1-7, characterized in that, The preparation method includes mixing Akkermansia muciniphila, milk thistle extract and artichoke extract in parts by weight.

9. A functional food or health product, characterized in that, The functional food or health product described herein comprises the liver-protecting composition according to any one of claims 1-7.

10. The functional food or health product according to claim 9, characterized in that, The dosage forms of the functional foods or health products mentioned include liquid dosage forms, solid dosage forms, or semi-solid dosage forms.

11. The functional food or health product according to claim 9, characterized in that, The functional foods or health products mentioned also include nutritionally acceptable nutrients.

12. A medicine, characterized in that, The medicine comprises the liver-protective composition according to any one of claims 1-7.

13. The pharmaceutical product according to claim 12, characterized in that, The dosage forms of the medicine include any one or more of the following: tablets, pills, powders, suspensions, gels, emulsions, creams, granules, capsules, suppositories, injections, sprays, and injections.

14. The pharmaceutical product according to claim 12, characterized in that, The medicine also includes pharmaceutically acceptable excipients.

15. The use of the liver-protecting composition according to any one of claims 1-7, characterized in that, The applications include any one or more of the following: (1) in the preparation of functional foods or health products that have an auxiliary protective effect against chemically induced liver injury; (2) in the preparation of drugs for the prevention or treatment of liver injury.

16. The application according to claim 15, characterized in that, The drugs for preventing or treating liver injury include any one or more of the following: drugs for preventing or treating alcoholic liver injury, drugs for preventing or treating chemically induced liver injury, drugs for preventing or treating non-alcoholic fatty liver injury, and drugs for preventing or treating autoimmune liver injury.

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