Health-care food composition for assisting in protecting chemical liver injury and preparation method of health-care food composition

Abstract

The invention discloses a health-care food composition with an auxiliary protection function on chemical liver injury and a preparation method of the health-care food composition, and belongs to the technical field of health-care products. The health-care food composition with the auxiliary protection function on the chemical liver injury is prepared from radix paeoniae alba, fructus aurantii immaturus, honey-fried licorice root and folium mori, wherein the mass ratio of the radix paeoniae alba to the fructus aurantii immaturus to the honey-fried licorice root to the folium mori is 1: 1: 1: (1.5-2). The health-care food composition can effectively reduce the increase of transaminase in a chemical liver injury model and remarkably improve the form and inflammation phenomenon of the liver, has the effect of protecting the liver, has the same effect as the original formula of the Siji powder, and is a safe and low-toxicity health-care food composition capable of assisting in protecting the chemical liver injury.

Description

Technical Field

[0001] The invention relates to a health food composition with auxiliary protection against chemical liver damage and a preparation method thereof, belonging to the technical field of health products. Background Art

[0002] The liver is an important organ in the human body and plays a key role in human metabolism. It is involved in processes such as sugar metabolism and protein decomposition. Conventional drugs for treating liver disease are clinically effective, but side effects cannot be ignored, such as nausea, headache, allergies, etc. In addition, these drugs cannot play a preventive role, and the early stages of liver disease are difficult to detect because there are no obvious symptoms. By the time obvious symptoms are found during examination, the disease has entered a serious stage. Therefore, the development of preventive measures based on functional foods can fundamentally prevent liver damage.

[0003] Modern pharmacological studies have shown that the research and development of natural medicines driven by TCM theory is a feasible method for treating liver damage. Sini San originated from Zhang Zhongjing's Treatise on Febrile and Miscellaneous Diseases in the Han Dynasty and is a classic TCM prescription for treating liver and spleen diseases. Sini San is composed of Bupleurum, White Peony, Citrus aurantium, and Radix Glycyrrhizae in a ratio of 1:1:1:1. The whole prescription soothes the liver and strengthens the spleen, relieves depression and clears away heat, combines dispersion and collection, and has both ascending and descending effects. However, some of the Chinese medicinal raw materials involved are prohibited from being added to food due to their particularity, which limits the application of Chinese medicinal compound prescriptions in functional foods. Summary of the invention

[0004] [Technical issues]

[0005] Although Sini San has a positive therapeutic effect in soothing the liver and strengthening the spleen, some of the Chinese medicinal ingredients in its formula are prohibited from being added to food, which limits its application in daily functional foods and makes it impossible to prevent liver damage in daily life.

[0006] [Technical solution]

[0007] In view of the above-mentioned technical problems, the purpose of the present invention is to provide a health food composition with auxiliary protection against chemical liver damage and a preparation method thereof. Specifically, by analyzing the medicinal properties of some medicinal and edible materials, screening out those with functional properties similar to these Chinese medicine raw materials, and then confirming the formula dosage through network pharmacology analysis, finally, obtaining a health food composition with good auxiliary protection against chemical liver damage. The composition has good effect, safety and low toxicity, thereby guiding the development and research of functional foods.

[0008] In order to achieve the above purpose, the following technical solutions are provided:

[0009] The invention provides a health food composition with auxiliary protection against chemical liver damage. The health food composition consists of white peony root, immature bitter orange, roasted liquorice root and mulberry leaf, wherein the mass ratio of the white peony root, immature bitter orange, roasted liquorice root and mulberry leaf is 1:1:1:1.5-2.

[0010] In one embodiment, the mulberry leaves are dried leaves of the mulberry plant of the Moraceae family, which are harvested after the first frost, impurities are removed, and the leaves are dried in the sun.

[0011] In one embodiment, the mass ratio of white peony root, immature bitter orange, roasted liquorice root and mulberry leaf is 1:1:1:1.67.

[0012] In one embodiment, the health food composition consists of 1 part of white peony root, 1 part of immature bitter orange, 1 part of roasted liquorice root, and 1.67 parts of mulberry leaf.

[0013] The present invention also provides a method for preparing the above-mentioned freeze-dried powder of the health food composition having the function of assisting in protecting chemical liver damage, the preparation method comprising the following steps:

[0014] (1) crushing and grinding the raw materials of white peony root, immature bitter orange, roasted liquorice root and mulberry leaf for later use;

[0015] (2) Add the health food composition into a constant temperature heating device in accordance with the formula ratio, add water, soak and extract in water, centrifuge to obtain the supernatant, and freeze-dry to obtain a freeze-dried powder of the health food composition.

[0016] In one embodiment, the mass ratio of the water to the health food composition in step (2) is 10-15:1, preferably 10:1.

[0017] In one embodiment, the extraction in step (2) refers to soaking for 30 to 60 minutes, and then heating under reflux at 95 to 100° C. for 1 to 3 hours.

[0018] In one embodiment, the centrifugation conditions in step (2) are: 9000-12000×g, 4° C., 10-15 min.

[0019] In one embodiment, the freeze-drying conditions in step (2) are: the cold trap temperature is -20°C to -40°C, the vacuum degree is 0.01 to 0.05 KPa, and the drying time is 30h to 60h.

[0020] The present invention also provides a freeze-dried powder of a health food composition obtained by the above-mentioned preparation method.

[0021] The present invention also provides the use of the above-mentioned health food composition or the lyophilized powder of the health food composition in the preparation of a product for auxiliary protection of chemical liver damage.

[0022] In one embodiment, the product comprises any one of a solid particle beverage, a compressed buccal tablet, and a concentrated paste.

[0023] In one embodiment, the product is in the form of any one of soft capsules, pills, microcapsules, oral liquids or powders.

[0024] Beneficial effects:

[0025] (1) The health food composition with auxiliary protection against chemical liver injury of the present invention is obtained by reasonable replacement based on the original formula of Sini San. Sini San is a classic Chinese medicine prescription for treating liver and spleen. The whole prescription soothes the liver and strengthens the spleen, relieves depression and clears heat, combines dispersing and collecting, and has ascending and descending effects. Modern pharmacological studies have shown that Sini San has liver protection, lipid-lowering, anti-inflammatory, and antioxidant effects. Bupleurum in Sini San is the main ingredient of Sini San. Bupleurum is a herb that is considered bitter and cold in traditional Chinese medicine theory and belongs to the liver and gallbladder meridians. In Sini San, Bupleurum mainly plays an anti-inflammatory, liver-soothing and depression-relieving, immune regulation, and antioxidant effects. Mulberry leaf is a bitter and cold material that belongs to the liver and lung meridians. Modern pharmacological studies have shown that mulberry leaf has a wide range of pharmacological effects, covering antioxidant, anti-inflammatory, lipid-lowering, anti-tumor, liver protection, anti-allergic and other effects. Replacing Bupleurum with mulberry leaf meets the principle of similarity in nature and flavor and meridians, and can cover the effect of Bupleurum in terms of efficacy.

[0026] (2) The replacement effect was verified using a carbon tetrachloride-induced mouse liver injury model. The results showed that the health food composition can effectively reduce the increase of transaminase in the chemical liver injury model, significantly improve the morphology and inflammatory phenomena of the liver, and has the effect of protecting the liver. The health food composition has the same efficacy as the original formula of Sini San, and is a safe, low-toxic health food composition with auxiliary protection against chemical liver injury, which provides a new idea for the further development of functional foods with liver protective effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 IC of Bupleurum chinense water extract and Morus alba water extract 50 Value curve graph;

[0028] Figure 2 This is a data graph showing the effects of each treatment group on the liver index of mice with chemical liver injury in Example 3;

[0029] Figure 3 The actual pictures of liver autopsies of mice with chemical liver injury in each treatment group in Example 3;

[0030] Figure 4The data graphs show the effects of each treatment group in Example 3 on the serum indexes of mice with chemical liver injury; (A) ALT level; (B) AST level; (C) albumin content; (D) AKP activity; (E) TBA content;

[0031] Figure 5 The cell section diagram shows the effect of each treatment group in Example 3 on the liver tissue morphology of mice with chemical liver injury. DETAILED DESCRIPTION

[0032] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The following specific implementation methods further describe the present invention.

[0033] Sources of materials involved in the present invention:

[0034] HepG2 cells were purchased from the Cell Bank of the Chinese Academy of Sciences; DMEM culture medium was purchased from Gibco; fetal bovine serum was purchased from Lonsera in Uruguay; ALT and AST kits were purchased from Beijing Solebow Technology Co., Ltd.

[0035] Carbon tetrachloride (CCl4) and olive oil were purchased from Shanghai Titan Technology Co., Ltd.; silymarin was purchased from Sigma; paraformaldehyde (4%) was purchased from Anhui White Shark Biotechnology Co., Ltd.; alanine aminotransferase (ALT) and aspartate aminotransferase (AST) kits were purchased from Beijing Solebow Technology Co., Ltd. Albumin (ALB), alkaline phosphatase (AKP), and total bile acid (TBA) kits were purchased from Nanjing Jiancheng Bioengineering Institute.

[0036] The bupleurum in Sinisan (white peony root, immature bitter orange, roasted liquorice root, bupleurum) is a raw material that cannot be added to food. In traditional Chinese medicine research, bupleurum is a Chinese medicine with "cold nature and bitter taste". According to the principle of similarity in nature, taste and meridians, 16 medicinal and edible materials with similar nature, taste and meridians as bupleurum were screened from the Pharmacopoeia of the People's Republic of China; mulberry leaves with the highest coverage rate were selected as replacement materials based on the coverage of bupleurum target points.

[0037] Example 1: Substitution materials and dosage determination of Radix Bupleuri in the formula of Sini Powder

[0038] (1) Confirmation of the replacement material of Bupleurum in Sini San

[0039] According to the network pharmacology research method, the potential active targets of Bupleurum and 16 medicinal and edible materials with similar properties and meridians as Bupleurum were obtained respectively. At the same time, the disease targets of "chemical liver injury" were obtained in the database, and the disease targets were intersected with the active targets of each single substance to obtain the number of repeated targets. The targets of the 16 medicinal and edible materials were intersected with the intersection targets of Bupleurum again to obtain the number of repeated targets and replacement rate of each single drug and Bupleurum. According to the replacement rate, mulberry leaf with the highest replacement rate (90%) was selected as the proposed replacement material.

[0040] (2) Preparation of water extracts of Bupleurum and mulberry leaves

[0041] Weigh 10g of Bupleurum, add 10 times the amount of water, then add it to a constant temperature heating device, soak it in water for half an hour, heat it to 99±1℃ and reflux it for 1h, then centrifuge to take the supernatant, and repeat the extraction of the residue for 3 times; after mixing the supernatants of the three extractions, freeze-dry them for 50h at a cold trap temperature of -40℃ and a vacuum degree of 0.01KPa to obtain a lyophilized powder; the lyophilized powder is re-dissolved to obtain a Bupleurum water extract, and the yield of the water extract is the ratio of the mass of the lyophilized powder to the mass of the raw material.

[0042] The extraction process of mulberry leaf water extract is the same as that of Bupleurum.

[0043] (3) HepG2 cell experiments

[0044] HepG2 cells were cultured in DMEM containing 10% fetal bovine serum in a humidified incubator at 37°C and 5% CO2, and the cells were passaged every two days.

[0045] HepG2 cells (1×10 6 After culturing for 24 h, 0.6% carbon tetrachloride culture medium containing different concentrations of water extract (50-400 μg / mL) was added and cultured for 24 h. After the culture, the cell pellets were collected, and the cells were lysed with RIPA lysis buffer. The protein concentration was determined with a BCA kit, and the AST level was determined according to the instructions of the kit. The IC 50 value.

[0046] (4) Determine the amount of mulberry leaves to replace bupleurum

[0047] According to the water extract yield (N) of Bupleurum chinense and mulberry leaves and the inhibition rate of aspartate aminotransferase (AST), a specific indicator of liver damage, in cell experiments, the IC 50 Value (X), calculate and determine the replacement amount M of each alternative according to formula (1).

[0048]

[0049] Where: N1, mulberry leaf water extract yield; X1, mulberry leaf IC 50 value; N2, yield of water extract of Bupleurum chinense; X2, IC 50 value.

[0050] like Figure 1 As shown, the IC values ​​of water extracts of Bupleurum chinense and Morus alba were 50 The values ​​were 75.93 μg / mL and 143.40 μg / mL, respectively, and their yields were 23.30% and 26.30%, respectively. The replacement ratio was calculated according to formula (1). The calculation result showed that 1.67 parts of mulberry leaves replaced 1 part of bupleurum. After the replacement, the new formula was composed entirely of materials that can be used in health food (white peony root, immature bitter orange, roasted liquorice root, mulberry leaves). The new formula takes into account the traditional concept of Chinese medicine and ensures the integrity of the prescription.

[0051] Example 2

[0052] A method for preparing a health food composition freeze-dried powder (SYT), comprising the following steps:

[0053] (1) crushing and grinding the raw materials of white peony root, immature bitter orange, roasted liquorice root and mulberry leaf for later use;

[0054] (2) Add white peony root, immature bitter orange, roasted licorice root, and mulberry leaf according to the formula ratio (including 10 g each of white peony root, immature bitter orange, and roasted licorice root, and 16.7 g of mulberry leaf) into a constant temperature heating device, add distilled water with a volume ten times that of the medicinal materials, soak in water for half an hour, heat to 99±1°C and reflux for 1 hour, then centrifuge at 4°C and 9000×g for 10 minutes to obtain the supernatant, repeat the extraction of the residue three times, combine the supernatants, and freeze-dry for 50 hours at a cold trap temperature of -40°C and a vacuum degree of 0.01 KPa to obtain a lyophilized powder.

[0055] Comparative Example 1

[0056] The preparation of Sinisan freeze-dried powder (SNS) comprises the following steps:

[0057] (1) crushing and grinding the raw materials of white peony root, immature bitter orange, roasted liquorice root and radix bupleuri for later use;

[0058] (2) Add white peony root, immature bitter orange, roasted liquorice root and bupleurum root according to the formula ratio (10 g each) into a constant temperature heating device, add distilled water with a volume ten times that of the medicinal materials, soak in water for half an hour, heat to 99±1°C and reflux for 1 hour, then centrifuge at 4°C and 9000×g for 10 minutes to obtain the supernatant, repeat the extraction of the residue three times; combine the supernatants, freeze-dry for 50 hours at a cold trap temperature of -40°C and a vacuum degree of 0.01 KPa to obtain a lyophilized powder.

[0059] Example 3

[0060] Experimental study on the mouse model of chemical liver injury induced by carbon tetrachloride

[0061] Male 8-week-old C57BL / 6J mice were purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd. All mice were acclimated to a 12-h light / 12-h dark cycle at 22±2°C and 40-70% relative humidity with free access to water and food. The animal protocol was approved by the Animal Experiment Ethics Review Committee of Jiangnan University (approval number JN.No20240630c1600822

[347] ).

[0062] Male 8-week-old C57BL / 6J mice, 10 mice in each group, a total of 7 groups: blank control, model control, positive control (silymarin, 100 mg / kg), Example 2 low concentration group (3.0 g / kg), Example 2 high concentration group (6.0 g / kg), Comparative Example 1 low concentration group (3.0 g / kg), Comparative Example 1 high concentration group (6.0 g / kg).

[0063] This experiment chose to conduct drug administration and modeling at the same time, and the drug treatment cycle was the same as the modeling. Except for the blank group, the other groups were intraperitoneally injected with carbon tetrachloride olive oil (v:v, 1:49) solution twice a week with an injection dose of 5mL / kg to establish a carbon tetrachloride chemical liver injury model. All samples were dissolved in physiological saline and administered by gavage, with a dosage of 0.2mL once a day, and the experimental period was 5 weeks.

[0064] Results Analysis

[0065] 1. After the experiment, weigh the mouse liver and calculate the liver index.

[0066] The results are as follows Figure 2 As shown, compared with the normal group, the liver index of the carbon tetrachloride modeling group increased significantly to 129.0%, while after treatment with SYT, SNS and positive control samples, the liver index decreased significantly, among which high concentration of SYT treatment could reduce it to 85.5% of the model group, with the best reduction effect. There was no significant difference compared with the positive samples and Sinisan.

[0067] 2. After the experiment, the mouse liver was removed for autopsy and photographed with a camera.

[0068] The results are as follows Figure 3 As shown in the figure, the livers of mice in the blank group were smooth, delicate and shiny. After the carbon tetrachloride modeling treatment, it was found that the surface of the livers of mice in the model group was rough, granular, dull and matte, in sharp contrast to the blank group. After intervention with different sample groups, the livers of mice showed significant improvements. Both the SNS and SYT groups made the liver surface smoother and closer to normal liver tissue, and the livers of mice showed different degrees of improvement.

[0069] 3. Determination of mouse serum indicators

[0070] The mouse serum was taken to measure the alanine aminotransferase (ALT) activity, aspartate aminotransferase (AST) activity, albumin (ALB) content, alkaline phosphatase (AKP) activity, and total bile acid (TBA) content. The determination method was carried out according to the instructions of the kit.

[0071] The results are as follows Figure 4 As shown in the results, compared with the normal group, the serum indicators of the model group changed significantly. The ALT level, AST level, AKP activity, and TBA content of the model group increased to 807.4%, 417.1%, 160.3%, and 593.2% of the blank group, respectively, and the ALB content decreased to 71.5% of the blank group. Low-concentration and high-concentration SYT intervention can significantly improve the changes of these indicators, and there is a certain dose effect. High-concentration SYT can reduce the ALT level, AST level, AKP activity, and TBA content to 62.4%, 63.6%, 78.8%, and 70.4% of the model group, respectively, and increase the ALB content to 117.3% of the model group. It has a liver protective effect similar to that of Sinisan and the positive drug silymarin.

[0072] 4. Effects on mouse liver tissue morphology

[0073] Fresh liver tissues of mice were washed with PBS and fixed with 4% paraformaldehyde. After dehydration, paraffin embedding and sectioning, they were stained with hematoxylin and eosin (H&E) to observe the morphology of liver tissues.

[0074] The results are as follows Figure 5 As shown in the figure, after HE staining of liver sections of mice in the blank group, it was found that the hepatocytes had normal morphology and structure, were neatly arranged, had clear nuclei and uniform cytoplasm structure. However, in the model group damaged by carbon tetrachloride, the hepatocytes were arranged in disorder, and the liver tissue showed large areas of ballooning, necrosis and inflammation. After intervention with each sample, the necrosis phenomenon was significantly improved, and the arrangement of hepatocytes was closer to normal liver tissue.

[0075] The above results indicate that the health food composition SYT of Example 2 has a liver protection effect similar to that of the original formula of Sini San (SNS) prepared in Comparative Example 1, and is a health food composition with auxiliary protection against chemical liver damage.

[0076] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.

Claims

1. A health food composition having the function of assisting in protecting chemical liver damage, characterized in that: The health food composition consists of white peony root, immature bitter orange, roasted licorice root and mulberry leaf, wherein the mass ratio of the white peony root, immature bitter orange, roasted licorice root and mulberry leaf is 1:1:1:1.5-2.

2. The health food composition according to claim 1, characterized in that The mass ratio of white peony root, immature bitter orange, roasted liquorice root and mulberry leaf is 1:1:1:1.

67.

3. The health food composition according to claim 1, characterized in that The health food composition consists of 1 part of white peony root, 1 part of immature bitter orange, 1 part of roasted liquorice root and 1.67 parts of mulberry leaf.

4. A method for preparing a freeze-dried powder of the health food composition having auxiliary protection against chemical liver damage according to any one of claims 1 to 3, characterized in that: The preparation method comprises the following steps: (1) crushing and grinding the raw materials of white peony root, immature bitter orange, roasted liquorice root and mulberry leaf for later use; (2) Add the health food composition into a constant temperature heating device in accordance with the formula ratio, add water, soak and extract in water, centrifuge to obtain the supernatant, and freeze-dry to obtain a freeze-dried powder of the health food composition.

5. The method according to claim 4, characterized in that The mass ratio of water to the health food composition in step (2) is 10 to 15:

1.

6. The method according to claim 4, characterized in that The extraction conditions of step (2) are: soaking for 30 to 60 minutes, and then heating under reflux at 95 to 100° C. for 1 to 3 hours.

7. A freeze-dried powder of a health food composition prepared by the method according to any one of claims 4 to 6.

8. Use of the health food composition according to any one of claims 1 to 3 or the lyophilized powder of the health food composition according to claim 7 in the preparation of a product for assisting protection against chemical liver damage.

9. The use according to claim 8, characterized in that: The product comprises any one of a solid particle beverage, a compressed buccal tablet, and a concentrated paste.

10. The use according to claim 8, characterized in that: The product may be in the form of soft capsules, pills, microcapsules, oral liquids or powders.

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