A health food composition for assisting in the protection of chemical-induced liver damage and a method of preparing the same

Abstract

The application discloses a health food composition with auxiliary protection of chemical liver injury and a preparation method thereof, and belongs to the technical field of health products. The health food composition with auxiliary protection of chemical liver injury is composed of radix paeoniae alba, fructus aurantii, licorice and mulberry leaves, wherein the mass ratio of radix paeoniae alba, fructus aurantii, licorice and mulberry leaves is 1:1:1:1.5-2. The health food composition can effectively reduce the increase of transaminase in a chemical liver injury model, significantly improve the morphology and inflammation of the liver, has the effect of protecting the liver, has the same effect as the original prescription of Sini Powder, and is a health food composition with auxiliary protection of chemical liver injury, which is safe and low in toxicity.

Description

TECHNICAL FIELD

[0001] The present application relates to a health food composition with auxiliary protection of chemical liver injury and a preparation method thereof, and belongs to the technical field of health products. BACKGROUND

[0002] The liver is an important organ of the human body, and plays a key role in the metabolism of the human body. It participates in processes such as sugar metabolism and protein decomposition. Conventional drugs for treating the liver are clinically effective, but the side effects cannot be ignored, such as nausea, headache, allergy, etc. In addition, these drugs cannot play a preventive role, and the early stage of liver disease is not easy to be detected due to no obvious symptoms, and when obvious symptoms are found through examination, the disease has entered a serious stage. Therefore, the development of preventive means based on functional foods can fundamentally prevent liver damage.

[0003] Modern pharmacological studies have shown that natural medicine research and development driven by traditional Chinese medicine theory is a feasible method for treating liver damage. Sini San originated from Treatise on Febrile Diseases and Miscellaneous Diseases by Zhang Zhongjing in the Han Dynasty, and is a classic prescription for treating liver and spleen in traditional Chinese medicine. Sini San is composed of Radix Bupleuri, Radix Paeoniae Alba, Fructus Aurantii Immaturus and Radix Glycyrrhizae in a ratio of 1:1:1:1, and the whole prescription can soothe the liver, invigorate the spleen, resolve stagnation and clear heat, disperse and absorb, and ascend with descending. However, some traditional Chinese medicine raw materials involved therein are prohibited from being added into food due to their particularity, thereby limiting the application of traditional Chinese medicine compound in functional food. SUMMARY

[0004] [TECHNICAL PROBLEM]

[0005] Although Sini San has a positive effect on soothing the liver and invigorating the spleen, some traditional Chinese medicine raw materials in its formula are prohibited from being added into food, thereby limiting its application in daily functional food, and thus it is impossible to prevent liver damage from daily life.

[0006] [TECHNICAL SCHEME]

[0007] In view of the above technical problems, the present application aims to provide a health food composition with auxiliary protection of chemical liver injury and a preparation method thereof, specifically by analyzing the medicinal properties of some medicinal and edible materials, screening out those with similar functional properties to the traditional Chinese medicine raw materials, and then analyzing and confirming the formula and dosage through network pharmacology, finally obtaining a health food composition with good auxiliary protection of chemical liver injury. The composition has good effect and low toxicity, thereby guiding the development and research of functional food.

[0008] In order to achieve the above-mentioned purpose, the following technical scheme is provided:

[0009] The application provides a health food composition with auxiliary protection of chemical liver injury, which is composed of radix paeoniae alba, fructus aurantii, licorice and mulberry leaves, wherein the mass ratio of radix paeoniae alba, fructus aurantii, licorice and mulberry leaves is 1:1:1:1.5-2.

[0010] In an embodiment, the mulberry leaves are dry leaves of Morus alba, which are collected after the first frost, impurities are removed, and the obtained product is dried.

[0011] In an embodiment, the mass ratio of radix paeoniae alba, fructus aurantii, licorice and mulberry leaves is 1:1:1:1.67.

[0012] In an embodiment, the health food composition is composed of 1 part of radix paeoniae alba, 1 part of fructus aurantii, 1 part of licorice and 1.67 parts of mulberry leaves.

[0013] The application also provides a preparation method of the freeze-dried powder of the health food composition with auxiliary protection of chemical liver injury.

[0014] (1) The components of radix paeoniae alba, fructus aurantii, licorice and mulberry leaves are crushed and powdered for standby;

[0015] (2) The health food composition is added into a constant-temperature heating device according to the formula ratio, and water is added to soak and extract, the supernatant is obtained by centrifugation, and freeze-drying is performed to obtain the freeze-dried powder of the health food composition.

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

[0017] In an embodiment, the extraction in step (2) refers to soaking for 30-60 min, and then heating and refluxing at 95-100 ℃ for 1-3 h.

[0018] In an embodiment, the centrifugation condition in step (2) is 9000-12000xg, 4 ℃, 10-15 min.

[0019] In an embodiment, the freeze-drying condition in step (2) is that the cold trap temperature is-20 ℃ to-40 ℃, the vacuum degree is 0.01-0.05 KPa, and the drying time is 30 h-60 h.

[0020] The application also provides the freeze-dried powder of the health food composition obtained by the preparation method.

[0021] The application also provides the application of the health food composition or the freeze-dried powder of the health food composition in the preparation of a product for auxiliary protection of chemical liver injury.

[0022] In an embodiment, the product comprises any one of a solid granular beverage, a compressed lozenge, a concentrated paste.

[0023] In an embodiment, the product is in the form of any one of a soft capsule, a pill, a microcapsule, an oral liquid, or a powder.

[0024] Beneficial effects:

[0025] (1) The health food composition with auxiliary protection of chemical liver injury of the present application is obtained by reasonably replacing the original Sini San. Sini San is a classic prescription for treating liver and spleen in traditional Chinese medicine, which soothes liver and strengthens spleen, resolves depression and clears heat, disperses and absorbs, and rises with descending. Modern pharmacological studies have shown that Sini San has the effects of protecting liver, lowering lipid, anti-inflammatory, and antioxidant. Bupleurum in Sini San is the main component of Sini San, which is a medicinal material considered bitter and cold in traditional Chinese medicine theory, belonging to liver and gallbladder channels. In Sini San, Bupleurum mainly plays the roles of anti-inflammatory, soothing liver and resolving depression, immune regulation, and antioxidant. Mulberry leaf is a bitter and cold material, belonging to liver and lung channels. Modern pharmacological studies have shown that mulberry leaf has a wide range of pharmacological effects, including antioxidant, anti-inflammatory, lipid-lowering, anti-tumor, liver protection, and anti-allergic effects. Replacing Bupleurum with mulberry leaf meets the similar principles of nature, taste, and meridian, and can cover the effects of Bupleurum.

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

[0027] Figure 1 IC value curve diagram of water extract of Bupleurum and water extract of mulberry leaf; 50

[0028] Figure 2 Data diagram of the influence of each treatment group in Example 3 on liver index of chemical liver injury mice;

[0029] Figure 3 Actual picture of liver dissection of chemical liver injury mice in each treatment group in Example 3;

[0030] Figure 4 ​Data graph of the influence of each treatment group in Example 3 on the serum indicators of the chemically injured mice; (A) is the ALT level; (B) is the AST level; (C) is the albumin content; (D) is the AKP activity; (E) is the TBA content.

[0031] Figure 5 Cell section graph of the influence of each treatment group in Example 3 on the liver tissue morphology of the chemically injured mice. DETAILED DESCRIPTION

[0032] The technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application. The following specific embodiments further describe the present application.

[0033] The material sources involved in the present application are as follows:

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

[0035] Carbon tetrachloride (CCl4) and olive oil were purchased from Shanghai Titan Science and Technology Co., Ltd.; silymarin was purchased from the sigma company; paraformaldehyde (4%) was purchased from Anhui BaiShi Biological Technology Co., Ltd.; glutathione (ALT) and glutathione (AST) kits were purchased from Beijing Solaybao Technology Co., Ltd. Albumin (ALB), alkaline phosphatase (AKP), and total bile acid (TBA) kits were purchased from Nanjing Jiancheng Biological Engineering Institute.

[0036] Radix Bupleuri in Sini San (Radix Paeoniae Alba, Fructus Aurantii Immaturus, Radix Glycyrrhizae, Radix Bupleuri) is a raw material that cannot be added to food. In traditional Chinese medicine research, Radix Bupleuri is a "cold and bitter" traditional Chinese medicine. According to the similarity principle of nature, taste and meridian, 16 food-medicinal materials with similar nature, taste and meridian to Radix Bupleuri were screened from the Pharmacopoeia of the People's Republic of China; according to the coverage of Radix Bupleuri target, the highest coverage of Morus alba was selected as the replacement material.

[0037] Example 1 Determination of replacement material and dosage of Radix Bupleuri in Sini San formula

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

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

[0040] (2) Preparation of water extract of Bupleurum and mulberry leaf

[0041] 10g of Bupleurum was weighed, 10 times the amount of water was added, then it was added to a constant temperature heating device, soaked in water for half an hour, heated to 99±1℃ reflux for 1h, then centrifuged to take the supernatant, the residue was extracted for 3 times; the supernatant of the three times of extraction was mixed, and then freeze-dried under the condition of cold trap temperature-40℃, vacuum degree 0.01KPa for 50h to obtain freeze-dried powder; the freeze-dried powder was redissolved to obtain Bupleurum water extract, and the yield of water extract was the ratio of the mass of freeze-dried powder to the mass of raw material.

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

[0043] (3) HepG2 cell experiment

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

[0045] HepG2 cells (1×10 6 per well) were inoculated in a 6-well plate and cultured for 24h, then 0.6% carbon tetrachloride culture solution containing different concentrations of water extract (50-400μg / mL) was added for co-culture for 24h, after the culture was completed, the cell precipitate was collected, the cells were lysed with RIPA lysis buffer, the protein concentration was determined by BCA kit, the AST level was determined according to the kit instructions, and the IC 50 value was determined according to the effect of different concentrations of water extract on reducing AST level.

[0046] (4) Determination of the dosage of mulberry leaf replacing Bupleurum

[0047] According to the water extract yield (N) of Bupleurum and mulberry leaf and the inhibition rate IC 50 value of the increase of aspartate aminotransferase (AST), a liver injury specific indicator in cell experiments, the replacement dosage M of each replacement formula was calculated according to formula (1).

[0048]

[0049] In the formula: N1, yield of mulberry leaf water extract; X1, IC50 of mulberry leaf extract. 50 Value; N2, yield of Bupleurum chinense water extract; X2, IC50 of Bupleurum chinense. 50 value.

[0050] like Figure 1 As shown, the water extracts of Bupleurum chinense and mulberry leaves have IC50 values. 50 The values ​​were 75.93 μg / mL and 143.40 μg / mL, respectively, and their yields were 23.30% and 26.30%, respectively. According to formula (1), the replacement ratio was calculated, and the result was 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, prepared licorice root, and mulberry leaves). The new formula takes into account the traditional Chinese medicine concept of medication and ensures the integrity of the prescription.

[0051] Example 2

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

[0053] (1) Crush and grind the raw materials of white peony root, immature bitter orange, prepared licorice root and mulberry leaf into powder for later use;

[0054] (2) Add white peony root, immature bitter orange, prepared licorice root, and mulberry leaf to a constant temperature heating device according to the formula ratio (10g each of white peony root, immature bitter orange, and prepared licorice root, and 16.7g of mulberry leaf), add ten times the volume of distilled water to the medicinal materials, soak in water for half an hour, heat to 99±1℃ and reflux for 1 hour, then centrifuge at 4℃ and 9000×g for 10 minutes to collect the supernatant. Repeat the extraction of the residue 3 times, combine the supernatant, and freeze dry at a cold trap temperature of -40℃ and a vacuum degree of 0.01KPa for 50 hours to obtain freeze-dried powder.

[0055] Comparative Example 1

[0056] The preparation of Si Ni San lyophilized powder (SNS) includes the following steps:

[0057] (1) Crush and grind the raw materials of white peony root, immature bitter orange, prepared licorice root and bupleurum root into powder for later use;

[0058] (2) Add white peony root, immature bitter orange, prepared licorice root and bupleurum root to a constant temperature heating device according to the formula ratio (10g each), add ten times the volume of distilled water, soak in water for half an hour, heat to 99±1℃ and reflux for 1h, then centrifuge at 4℃ and 9000×g for 10min to collect the supernatant, and repeat the extraction of the residue 3 times; combine the supernatants, freeze dry at a cold trap temperature of -40℃ and a vacuum degree of 0.01KPa for 50h to obtain freeze-dried powder.

[0059] Example 3

[0060] Carbon tetrachloride-induced chemical liver injury mouse model experiment

[0061] Male 8-week-old C57BL / 6J mice were purchased from Zhejiang Vantong Lihua Experimental Animal Technology Co., Ltd. All mice were adapted to a 12-hour light / 12-hour dark cycle at 22 ± 2°C and 40-70% relative humidity, with free access to water and food. The animal program was approved by the Animal Experiment Ethics Review Committee of Jiangnan University (Approval No. JN.No20240630c1600822

[347] ).

[0062] Male 8-week-old C57BL / 6J mice, 10 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] In this experiment, drug administration was performed at the same time as modeling, and the treatment period of the drug was the same as the modeling period. Except for the blank group, the remaining groups were intraperitoneally injected with carbon tetrachloride olive oil (v:v, 1:49) solution, twice a week, with an injection dose of 5 mL / kg, to establish a carbon tetrachloride chemical liver injury model. All samples were dissolved in normal saline and administered by gavage, with a dosage of 0.2 mL once a day, and the experimental period was 5 weeks.

[0064] Results analysis

[0065] 1. After the experiment, the mouse livers were weighed and the liver index was calculated.

[0066] The results are shown in Table 1. Figure 2 As shown in Table 1, 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, and the high concentration of SYT treatment could reduce to 85.5% of the model group, with the best reduction effect, and no significant difference compared with the positive sample and Sini Powder.

[0067] 2. After the experiment, the mouse livers were dissected and photographed with a camera.

[0068] The results are shown in Table 2. Figure 3 As shown in Table 2, the livers of the mice in the blank group were smooth, delicate, and shiny. After treatment with carbon tetrachloride, it was found that the livers of the mice in the model group were rough, with a grainy feel and a dull, non-shiny appearance, which was in sharp contrast to the blank group. After intervention with different sample groups, the livers of the mice improved significantly, and SNS and SYT groups could make the liver surface smoother and more similar to normal liver tissue, with different degrees of improvement in the livers of the mice.

[0069] 3. Determination of mouse serum indexes

[0070] The mouse serum was taken to determine the activities of glutamic-pyruvic transaminase (ALT), glutamic-oxaloacetic transaminase (AST), the content of albumin (ALB), the activity of alkaline phosphatase (AKP), and the content of total bile acid (TBA), and the determination methods were performed according to the instructions of the kits.

[0071] The results are shown in Table 2. Figure 4 Compared with the normal group, the serum indexes of the model group were significantly changed, and the ALT level, AST level, AKP activity, and TBA content of the model group were increased to 807.4%, 417.1%, 160.3%, and 593.2% of the blank group, respectively, and the ALB content was decreased to 71.5% of the blank group. The low and high concentrations of SYT could significantly improve the changes of these indexes, and there was a certain dose effect. The high concentration of SYT could 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. SYT had a similar liver protection effect as Sini Powder and the positive drug silymarin.

[0072] 4. Effect on the morphology of mouse liver tissue

[0073] The fresh liver tissue of the mouse was washed with PBS and fixed with 4% paraformaldehyde, and after dehydration, paraffin embedding, and sectioning, the liver tissue morphology was observed by hematoxylin and eosin (H&E) staining.

[0074] The results are shown in Table 3. Figure 5 After HE staining of the liver sections of the blank group mice, it was found that the hepatocyte morphology was normal, arranged in order, and had clear nucleus and uniform cytoplasm structure. The model group damaged by carbon tetrachloride showed disordered arrangement of hepatocytes, and the liver tissue showed a large area of balloon-like degeneration, necrosis, and inflammation. After the intervention of each sample, the necrosis was significantly improved, and the arrangement of hepatocytes was closer to that of normal liver tissue.

[0075] The above results show that the health food composition SYT of Example 2 has a similar liver protection effect as Sini Powder (SNS) prepared in Comparative Example 1, and is a health food composition for assisting in the protection of chemical liver damage.

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

Claims

1. A health food composition with auxiliary protective effect against chemically induced liver damage, characterized in that, The health food composition is composed of white peony root, bitter orange fruit, honey-fried licorice, and mulberry leaves, wherein the mass ratio of white peony root, bitter orange fruit, honey-fried licorice, and mulberry leaves is 1:1:1:1.

67.

2. The health food composition according to claim 1, characterized by, The health food composition is composed of 1 part of white peony root, 1 part of bitter orange fruit, 1 part of honey-fried licorice, and 1.67 parts of mulberry leaves.

3. A process for preparing a lyophilized powder of the health food composition having an auxiliary protective effect on chemical-induced liver injury according to any one of claims 1 or 2, characterized by, The preparation method comprises the following steps: (1) crushing and powdering each component raw material of white peony root, bitter orange fruit, honey-fried licorice, and mulberry leaves for standby; (2) adding the health food composition formula proportion into a constant temperature heating device, adding water, soaking and extracting in the water, centrifuging to obtain supernatant, and freeze-drying to obtain the health food composition freeze-dried powder.

4. The method of claim 3, wherein, In step (2), the mass ratio of water to the health food composition is 10-15:

1.

5. The method of claim 3, wherein, In step (2), the extraction condition is: soaking for 30-60 min, and then heating and refluxing at 95-100 ℃ for 1-3 h.

6. The health food composition freeze-dried powder obtained by the method of any one of claims 3-5.

7. The use of the health food composition of any one of claims 1 or 2 or the health food composition freeze-dried powder of claim 6 in the preparation of a product for assisting in protecting chemical liver injury.

8. Use according to claim 7, characterized in that, The product comprises any one of a solid granular beverage, a compressed tablet, and a concentrated paste.

9. Use according to claim 7, characterized in that, The form of the product comprises any one of a soft capsule, a pill, a microcapsule, an oral liquid, or a powder.

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