A composition for protecting alcoholic liver injury, a preparation method thereof, and an application thereof

Abstract

The application discloses a composition for protecting alcoholic liver injury, a preparation method and application thereof. The preparation raw materials of the composition comprise the following components in parts by weight: 1-6 parts of peony seed oil, 1-8 parts of radix puerariae, 1-4 parts of hovenia dulcis thunb, and 1-2 parts of gardenia. The four components are matched with each other and jointly act, have the effects of improving the body antioxidant capacity and reducing the fat deposition in liver cells, and have good effects on preventing and inhibiting alcoholic liver injury. Research proves that the composition has definite effects, no toxic and side effects, and has wide clinical application prospects.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant extracts, and particularly relates to a composition for protecting alcoholic liver injury, a preparation method and application thereof. BACKGROUND

[0002] With the improvement of living standards, changes in dietary structure and the increasing consumption of alcohol, the incidence of alcoholic liver disease (ALD) in China has shown an increasing trend year by year, which has caused systematic harm to the health of the Chinese population. ALD is related to many factors, among which oxidative stress generated during ethanol metabolism and lipid peroxidation damage caused thereby are the most important causes of ALD. At the same time, low-level intestinal endotoxemia induced by endogenous toxins causes inflammation, necrosis and fibrosis of fatty liver, thereby causing secondary damage to the liver, which has also been considered as another important mechanism of ALD in recent years.

[0003] In addition to alcohol abstinence and nutritional support, drug therapy is the main treatment for ALD. Although the currently widely used antioxidant drugs glutathione and silymarin can eliminate the oxidative stress caused by alcohol, they cannot effectively improve the symptoms of alcoholic fatty liver inflammation. Glucocorticoids may be the most effective drug for severe ALD, but the mortality rate of patients receiving hormone therapy is still high, and its effect on liver cirrhosis progression and improvement of long-term survival rate is not clear. Therefore, it is particularly urgent and important to explore safer and more effective intervention means for preventing and treating ALD. SUMMARY

[0004] In view of the deficiencies of the prior art, the present application provides a composition for protecting alcoholic liver injury, a preparation method and application thereof.

[0005] In one aspect of the present application, a composition for protecting alcoholic liver injury is provided, and the preparation raw materials thereof include the following components in parts by weight: 1-6 parts of peony seed oil, 1-8 parts of pueraria, 1-4 parts of jujube fruit, and 1-2 parts of mountain ziziphus. Preferably, the preparation raw materials thereof include the following components in parts by weight: 3 parts of peony seed oil, 4 parts of pueraria, 2 parts of jujube fruit, and 1 part of mountain ziziphus.

[0006] Another aspect of the present application provides a preparation method of the aforementioned composition for protecting liver injury caused by alcohol, comprising: S1, taking the mature fruit of Tribulus terrestris Linn, adding water to extract volatile oil, and reserving; preferably, the ratio of material to liquid is 1:4-6, and the extraction time is 3-5 h; more preferably, the ratio of material to liquid is 1:5, and the extraction time is 4 h; S2, mixing the residue after extracting volatile oil with Pueraria lobata and Radix Rubi, adding ethanol to extract, collecting the extract and recovering ethanol, and then concentrating, drying, and crushing to obtain dry powder of extract; preferably, the ratio of material to liquid is 1:6-10, the concentration of ethanol is 70-90% v / v, and the extraction time is 2-3 h; more preferably, the ratio of material to liquid is 1:8, the concentration of ethanol is 80% v / v, and the extraction time is 2.5 h; S3, combining the seed oil of Paeonia suffruticosa, volatile oil, and dry powder of extract, and obtaining the composition.

[0007] Another aspect of the present application provides a soft capsule of seed oil of Paeonia suffruticosa, which is composed of content and capsule material, and the preparation raw materials of the content include the following components in parts by weight: 1-6 parts of seed oil of Paeonia suffruticosa, 1-8 parts of Pueraria lobata, 1-4 parts of mature fruit of Tribulus terrestris Linn, 1-2 parts of Radix Rubi, and 0.5-1 part of beeswax; and the preparation raw materials of the capsule material include the following components in parts by weight: 1-1.1 parts of gelatin, 0.4-0.6 parts of glycerol, and 1-1.1 parts of water.

[0008] Another aspect of the present application provides a preparation method of the aforementioned soft capsule of seed oil of Paeonia suffruticosa, comprising:

[0009] a. preparing the content:

[0010] a1) taking the mature fruit of Tribulus terrestris Linn, adding water to extract volatile oil at a ratio of material to liquid of 1:4-6 for 3-5 h, and reserving;

[0011] a2) mixing the residue after extracting volatile oil with Pueraria lobata and Radix Rubi, adding ethanol to extract at a ratio of material to liquid of 1:6-10 for 2-3 h, collecting the extract and recovering ethanol, and then concentrating, drying, and crushing to obtain dry powder of extract;

[0012] a3) taking seed oil of Paeonia suffruticosa and beeswax, heating and melting, cooling to room temperature, adding dry powder of extract and volatile oil, and grinding uniformly to obtain the content;

[0013] b. preparing the capsule material: mixing gelatin, glycerol, and water, heating, and then cooling to form a gelatin skin;

[0014] c. pressing: wrapping the content with the gelatin skin and pressing to obtain the soft capsule.

[0015] The present application has the following beneficial effects:

[0016] The composition provided by the present application is prepared by taking peony seed oil, radix puerariae, hovenia dulcis thunb and gardenia as main raw materials, wherein 1) the peony seed oil is rich in alpha-linolenic acid, linoleic acid, oleic acid, vitamin E, squalene and polyphenol and other antioxidant substances. Alpha-linolenic acid and its metabolites can inhibit platelet aggregation by reducing the concentration of platelets to reduce the formation of thrombus, and have a significant effect on reducing blood lipids and preventing coronary heart disease. Linoleic acid can inhibit the synthesis of cholesterol, prevent cardiovascular diseases and senile obesity, and the regulation effect of oleic acid on low-density cholesterol (LDL) and high-density cholesterol (HDL) is selective, which can reduce LDL with large viscosity and easy to adsorb on the blood vessel wall, and increase the content of HDL with the effect of cleaning the LDL adhered to the blood vessel wall. Vitamin E and squalene have the physiological functions of increasing the activity of superoxide dismutase (SOD) in the body, enhancing the immunity of the body and resisting aging. Polyphenol can effectively reduce the damage of the body caused by oxidative stress and prevent various diseases related to free radical oxidation. 2) Radix puerariae has the effects of relieving muscle and fever, promoting rash, producing saliva and stopping thirst, raising yang and stopping diarrhea. It can improve the regeneration capacity of liver cells, restore normal liver function, promote bile secretion, prevent fat accumulation in the liver, promote metabolism, strengthen the detoxification function of the liver and prevent the damage of alcohol to the liver. 3) Hovenia dulcis thunb has the effects of promoting water and reducing swelling and removing alcohol toxicity. 4) Gardenia has the effects of removing heat and dampness, soothing liver and promoting bile, and has the effect of inhibiting inflammatory reaction. The above four components cooperate with each other and jointly act on the main effect of inhibiting oxidative stress and lipid peroxidation damage, and also consider anti-inflammatory, effectively inhibit the secondary damage of the liver caused by endogenous toxin-induced inflammatory reaction, and can significantly improve the symptoms of alcoholic liver damage, including abnormal liver function and fatty liver disease, and has no toxic side effects. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 It is the content extraction process of the peony seed oil soft capsule of the present application example 1;

[0018] Figure 2 It is the preparation process of the peony seed oil soft capsule of the present application example 1;

[0019] Figure 3 It is the analysis result of the influence of the peony seed oil soft capsule of the present application example 1 on MDA in the liver of acute alcoholic liver injury rats;

[0020] Figure 4 It is the analysis result of the influence of the peony seed oil soft capsule of the present application example 1 on GSH in the liver of acute alcoholic liver injury rats;

[0021] Figure 5 It is the analysis result of the influence of the peony seed oil soft capsule of the present application example 1 on TG in the liver of acute alcoholic liver injury rats;

[0022] Figure 6The influence analysis result of the peony seed oil soft capsule of the embodiment 1 of the present application on the liver pathological score of the acute alcoholic liver injury rat;

[0023] Figure 7 The influence analysis result of the peony seed oil soft capsule of the embodiment 1 of the present application on the MDA in the liver of the chronic alcoholic liver injury rat;

[0024] Figure 8 The influence analysis result of the peony seed oil soft capsule of the embodiment 1 of the present application on the GSH in the liver of the chronic alcoholic liver injury rat;

[0025] Figure 9 The influence analysis result of the peony seed oil soft capsule of the embodiment 1 of the present application on the SOD in the liver of the chronic alcoholic liver injury rat;

[0026] Figure 10 The influence analysis result of the peony seed oil soft capsule of the embodiment 1 of the present application on the TG in the liver of the chronic alcoholic liver injury rat;

[0027] Figure 11 The influence analysis result of the peony seed oil soft capsule of the embodiment 1 of the present application on the AST in the serum of the chronic alcoholic liver injury rat;

[0028] Figure 12 The influence analysis result of the peony seed oil soft capsule of the embodiment 1 of the present application on the ALT in the serum of the chronic alcoholic liver injury rat;

[0029] Figure 13 The influence analysis result of the peony seed oil soft capsule of the embodiment 1 of the present application on the liver index of the chronic alcoholic liver injury rat;

[0030] Figure 14 The influence analysis result of the peony seed oil soft capsule of the embodiment 1 of the present application on the liver pathological score of the chronic alcoholic liver injury rat;

[0031] Figure 15 is the influence analysis result of the peony seed oil soft capsule of the embodiment 1 of the present application on the pathological change of the liver of the acute alcoholic liver injury rat (HE staining, 400 times);

[0032] Figure 15A The influence analysis result of the normal group on the pathological change of the liver of the acute alcoholic liver injury rat (HE staining, 400 times);

[0033] Figure 15B The influence analysis result of the model group on the pathological change of the liver of the acute alcoholic liver injury rat (HE staining, 400 times);

[0034] Figure 15CThe influence analysis result (HE staining, 400 times) of the high-dose group of the peony seed oil soft capsule of the present application on the pathological changes of the liver of the acute alcoholic liver injury rats;

[0035] Figure 15D The influence analysis result (HE staining, 400 times) of the middle-dose group of the peony seed oil soft capsule of the present application on the pathological changes of the liver of the acute alcoholic liver injury rats;

[0036] Figure 15E The influence analysis result (HE staining, 400 times) of the low-dose group of the peony seed oil soft capsule of the present application on the pathological changes of the liver of the acute alcoholic liver injury rats;

[0037] Figure 16A The influence analysis result (HE staining, 400 times) of the normal group on the pathological changes of the liver of the chronic alcoholic liver injury rats;

[0038] Figure 16B The influence analysis result (HE staining, 400 times) of the model group on the pathological changes of the liver of the chronic alcoholic liver injury rats;

[0039] Figure 16C The influence analysis result (HE staining, 400 times) of the high-dose group of the peony seed oil soft capsule of the present application on the pathological changes of the liver of the chronic alcoholic liver injury rats;

[0040] Figure 16D The influence analysis result (HE staining, 400 times) of the middle-dose group of the peony seed oil soft capsule of the present application on the pathological changes of the liver of the chronic alcoholic liver injury rats;

[0041] Figure 16E The influence analysis result (HE staining, 400 times) of the low-dose group of the peony seed oil soft capsule of the present application on the pathological changes of the liver of the chronic alcoholic liver injury rats;

[0042] Figure 17 The pathological changes (HE staining, 400 times) of the liver tissues of the rats in the sample A and sample B groups. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical scheme and advantages of the embodiments of the present application more clear, the technical scheme of the embodiments of the present application will be clearly and completely described below with reference to the drawings of the embodiments of the present application. Obviously, the described embodiments are some of the embodiments of the present application, but not all the embodiments of the present application. 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.

[0044] Embodiment 1

[0045] A peony seed oil soft capsule for protecting alcoholic liver injury, which is composed of a content and a capsule material, the content is prepared from the following raw materials: peony seed oil 180 g, pueraria 240 g, hovenia dulcis 120 g, gardenia 60 g, and beeswax 50 g; the capsule material is prepared from the following raw materials: gelatin 100 g, glycerol 40 g, and water 100 g; the preparation process is shown in Figures 1-2 The preparation method is as follows:

[0046] a. Preparation of the content: volatile oil is extracted from hovenia dulcis with 5 times the amount of water for 4 h, and the volatile oil is stored separately for later use. The residues and pueraria and gardenia are mixed, and then extracted with 80% v / v ethanol by heating and refluxing twice, with a solid-liquid ratio of 1:8 each time, the first extraction time being 1.5 h and the second extraction time being 1.0 h. The extract is recovered with ethanol, concentrated and dried into dry extract powder, which is pulverized into fine powder and sieved through a 100-mesh sieve to obtain the dry extract powder. The peony seed oil and beeswax are heated and melted, and then cooled to room temperature. The dry extract powder and the volatile oil are added, and then uniformly ground in a colloid mill to obtain the content;

[0047] b. Preparation of the capsule material: gelatin, glycerol and water are mixed and heated, and then cooled to form a gelatin coating;

[0048] c. Compression: the content is wrapped with the gelatin coating, and then compressed into 1000 soft capsules, each capsule being 0.65 g. The serving method is 2 capsules each time, 3 times a day.

[0049] Example 2

[0050] A peony seed oil soft capsule for protecting alcoholic liver injury, which is composed of a content and a capsule material, the content is prepared from the following raw materials: peony seed oil 180 g, pueraria 240 g, hovenia dulcis 120 g, gardenia 60 g, and beeswax 50 g; the capsule material is prepared from the following raw materials: gelatin 100 g, glycerol 40 g, and water 100 g;

[0051] The preparation method is as follows:

[0052] a. Preparation of the content: volatile oil is extracted from hovenia dulcis with 5 times the amount of water for 4 h, and the volatile oil is stored separately for later use. The residues and pueraria and gardenia are mixed, and then extracted with 80% v / v ethanol by heating and refluxing twice, with a solid-liquid ratio of 1:8 each time, the first extraction time being 1.5 h and the second extraction time being 1.0 h. The extract is recovered with ethanol, concentrated and dried into dry extract powder, which is pulverized into fine powder and sieved through a 100-mesh sieve to obtain the dry extract powder. The peony seed oil and beeswax are heated and melted, and then cooled to room temperature. The dry extract powder and the volatile oil are added, and then uniformly ground in a colloid mill to obtain the content;

[0053] b. Preparation of the capsule material: gelatin, glycerol and water are mixed and heated, and then cooled to form a gelatin coating;

[0054] c. Compression: after the content is wrapped with the gelatin, the soft capsules are compressed to obtain 1000 capsules, each capsule being 0.65 g. The method of taking is 2 capsules each time, 3 times a day.

[0055] Example 3

[0056] A soft capsule for protecting alcoholic liver injury is composed of a content and a capsule material. The content is prepared from the following raw materials: 180 g of peony seed oil, 240 g of pueraria, 120 g of hovenia dulcis thunb, 60 g of gardenia, and 50 g of beeswax; and the capsule material is prepared from the following raw materials: 100 g of gelatin, 40 g of glycerol, and 100 g of water.

[0057] The preparation method is as follows:

[0058] a. Preparation of the content: volatile oil is extracted from hovenia dulcis thunb by adding 6 times the amount of water for 5 h, and the volatile oil is stored in another container for standby. The residues and pueraria and gardenia are mixed, and 90% v / v ethanol is added to heat and reflux extraction twice. The material-liquid ratio is 1:10, the first extraction time is 2.0 h, and the second extraction time is 1.0 h. The extraction liquid is recovered by ethanol, concentrated and dried into dry extract powder, which is pulverized into fine powder and sieved through a 100-mesh sieve to obtain the dry extract powder. The peony seed oil and beeswax are heated and melted, cooled to room temperature, and then the dry extract powder and volatile oil are added and uniformly ground in a colloid mill to obtain the content;

[0059] b. Preparation of the capsule material: the gelatin, glycerol and water are mixed and heated, and then the gelatin is cooled to form the gelatin.

[0060] c. Compression: after the content is wrapped with the gelatin, the soft capsules are compressed to obtain 1000 capsules, each capsule being 0.65 g. The method of taking is 2 capsules each time, 3 times a day.

[0061] Test example

[0062] (1) Prescription screening test research

[0063] The present application is based on the theory of traditional Chinese medicine, and the functions of various substances are screened and verified. It is clear that the selected peony seed oil+pueraria+hovenia dulcis thunb+gardenia formula has a protective effect on acute and chronic alcoholic liver injury in rats, thereby further providing a theoretical basis for the development of a function product for protecting alcoholic liver injury. The following is a part of the screening test research.

[0064] 1. Dose selection and administration method of the test substance

[0065] 1.1 Preparation of the extract

[0066] Preparation of Pueraria extract: Take Pueraria, add 80% v / v ethanol and heat to reflux for 2 times, each time with a material to liquid ratio of 1:8, the first time for 1.5 h and the second time for 1.0 h. Recover the ethanol from the extract, concentrate and dry to a dry extract, and pulverize to a fine powder.

[0067] Preparation of Hovenia extract: Take Hovenia, add 5 times the amount of water to extract the volatile oil for 4 h. Separate the volatile oil and store it in another container for later use. Add 80% v / v ethanol to the residue and heat to reflux for 2 times, each time with a material to liquid ratio of 1:8, the first time for 1.5 h and the second time for 1.0 h. Recover the ethanol from the extract, concentrate and dry to a dry extract, and pulverize to a fine powder. Combine the volatile oil and the dry extract powder to obtain the Hovenia extract.

[0068] Preparation of Gardenia extract: Take Gardenia, add 80% v / v ethanol and heat to reflux for 2 times, each time with a material to liquid ratio of 1:8, the first time for 1.5 h and the second time for 1.0 h. Recover the ethanol from the extract, concentrate and dry to a dry extract, and pulverize to a fine powder.

[0069] 1.2 Preparation of sample solution

[0070] Pueraria extract: 1 g is equivalent to 6.82 g of crude drug, Hovenia extract: 1 g is equivalent to 42.44 g of crude drug, and Gardenia extract: 1 g is equivalent to 6.67 g of crude drug.

[0071] Sample A: Take 5.86 g of Pueraria extract, add 0.47 g of Hovenia extract, add 1.5 g of Gardenia extract, dissolve in 10.6 ml of distilled water (maximum solubility), then add 30 g (32.33 ml) of peony seed oil and 4 ml of Tween 80 to make a total volume of 47 ml of stock solution.

[0072] Sample B: Take 5.86 g of Pueraria extract, add 0.7 g of Hovenia extract, dissolve in 10.6 ml of distilled water (maximum solubility), then add 30 g (32.33 ml) of peony seed oil and 4 ml of Tween 80 to make a total volume of 47 ml of stock solution.

[0073] 1.3 Calculation of sample dosage

[0074] The dosage of the entire prescription is calculated based on the daily dosage of peony seed oil. The daily dosage of peony seed oil for humans is 8 ml (6-10 ml), and the human dosage is 0.133 ml / kg. The high, medium and low doses for rats are set at 10 times, 5 times and 2.5 times, respectively. The calculated high, medium and low doses of the sample (peony seed oil) are 1.33, 0.67 and 0.33 ml / kg, respectively. Based on the fact that 47 ml of the sample contains 32.33 ml of peony seed oil, the calculated high, medium and low doses of the sample (peony seed oil) are 1.33, 0.67 and 0.33 ml / kg, respectively.

[0075] Sample A / B high, medium, low doses are 1.93, 0.96, 0.48 ml / kg of the original solution, and the required concentration is dissolved in distilled water before use.

[0076] Each test sample is set up with high, medium and low dose groups, and a normal control group and a model control group. The liver injury model is caused by anhydrous ethanol (analytical pure). The concentration of anhydrous ethanol is 50% (diluted with distilled water), and the intragastric volume is 12 mL / kg BW (equivalent to 6000 mg / kg BW of ethanol). The test sample is given for 30 days. The test sample is given orally by gavage, and the volume of the test sample is 1 ml / 100g of body weight.

[0077] The experimental method is as follows: SD rats weighing 180-220 g, male. Randomly divided into 8 groups, namely A high, medium, low, B high, medium, low, normal group, model group, 10 in each group. Each day, the test sample is given orally by gavage, and the normal group and the model control group are given distilled water. The animals are weighed twice a week, and the dose of the test sample is adjusted according to the body weight. At the end of the test sample administration, the model control group and each sample group are given 50% ethanol 12 mL / kg BW by gavage, and the normal control group is given distilled water. The animals are fasted for 16 hours and then sacrificed for detection of various indicators and observation of differences between groups.

[0078] Detection index 3.1: Take the liver of the animal and make homogenate. Detect the content of malondialdehyde (MDA) and reduced glutathione (GSH) in liver tissue by ultraviolet spectrophotometer, and measure the content of triglyceride (TG) by automatic biochemical analyzer.

[0079] 3.2 Pathological examination

[0080] Take samples from the middle of the left lobe of the liver, make frozen sections, and perform HE staining. Record the pathological changes of cells from the field of view of the liver, and observe the entire tissue section continuously using a 40x objective lens. The distribution, range and area of lipid droplets in the liver are mainly observed.

[0081] Pathological scoring criteria:

[0082]

[0083] 4. Result evaluation criteria: if any of the following conditions is met, the test sample is determined to have a protective effect on alcoholic liver injury:

[0084] (1) The results of MDA, reduced GSH and TG in liver are all positive (there is a statistical difference between the sample group and the model group)

[0085] (2) Two of the three indicators of MDA, reduced GSH and TG in liver are positive, and the result of pathological examination is positive.

[0086] 5. Experimental results

[0087] 5.1 Effect of each sample on MDA content in liver tissue

[0088] Compared with the normal group, the MDA content in the liver tissue of the model group animals was significantly increased, and the difference was significant. Compared with the model group, the high-, medium-, and low-dose sample A groups could significantly reduce the MDA content; the high-, medium-, and low-dose sample B groups could also significantly reduce the MDA content. Among them, the high-dose sample A group was more effective. The specific results are shown in Table 1.

[0089]

[0090] 5.2 Effect of each sample on GSH content in liver tissue

[0091] Compared with the normal group, the GSH content in the model group was significantly reduced, and the difference was significant. Compared with the model group, the high- and medium-dose sample A groups could significantly increase the GSH content, and the low-dose group had no obvious effect; the high- and medium-dose sample B groups could significantly increase the GSH content, and the low-dose group had no obvious effect. Among them, the high-dose sample A group was more effective. The specific results are shown in Table 2.

[0092]

[0093] 5.3 Effect of each sample on TG content in liver tissue

[0094] Compared with the normal group, the TG content in the liver tissue of the model group animals was significantly increased, and the difference was significant. Compared with the model group, the high-, medium-, and low-dose sample A groups could significantly reduce the TG content; the high-, medium-, and low-dose sample B groups could also significantly reduce the TG content. Among them, the high-dose sample A group was more effective in reducing TG. The specific results are shown in Table 3.

[0095]

[0096] 5.4 Pathological examination

[0097] The liver tissue structure of the normal group rats was normal, and there was no obvious lesion in the liver. There were scattered fat droplets in the liver cells.

[0098] The liver tissue cells of the model group rats contained a large number of fat droplets, and some appeared to be fused fat bubbles. Some liver cells were swollen, and there was more inflammatory cell infiltration.

[0099] The high-, medium-, and low-dose sample A / B groups could reduce the pathological changes of liver tissue to different degrees, and the high-dose group had a better effect on improving fatty degeneration, while the low-dose group had a relatively weak effect. Among them, the high-dose sample A group was more effective.

[0100] Liver histopathological score: Compared with the normal group, the liver histopathological score of the model group animals was significantly increased, and the difference was significant; compared with the model group, the high and medium dose groups of sample A can significantly reduce the pathological score; only the high dose group of sample B can significantly reduce the pathological score. Sample A is more obvious. See Table 4, Figure 17 .

[0101]

[0102] 6. Conclusion

[0103] 6.1 The auxiliary protection function of each sample on alcoholic liver injury has a dose-dependent effect.

[0104] 6.2 Sample A and B have positive results on MDA, GSH, TG and pathological detection results, indicating that the two samples have auxiliary protection function on alcoholic liver injury, but the high dose group of sample A is more obvious, so sample A is selected for further study.

[0105] (II) Test of the protective effect of the application on acute alcoholic liver injury

[0106] 1. Experimental animal grouping and treatment

[0107] SD rats weighing 180-220 g, half male and half female, were purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd. They were randomly divided into 5 groups, namely normal group, model group, peony seed oil soft capsule (prepared in Example 1) high, medium and low dose groups, 10 rats in each group. The test samples were given orally by gavage every day, and the normal group and the model control group were given distilled water. The animals were weighed twice a week, and the dose of the test sample was adjusted according to the body weight. At the end of the test sample administration, the model control group and each sample group were given 50% ethanol 12 mL / kg BW by gavage once, the normal control group was given distilled water, and the animals were sacrificed after fasting for 16 hours, and the corresponding indicators were detected to observe the differences between the groups.

[0108] 2. Index determination

[0109] 2.1 The liver of the animal was homogenized, and the content of malondialdehyde (MDA) and reduced glutathione (GSH) in the liver tissue was detected by ultraviolet spectrophotometer.

[0110] The content of triglyceride (TG) was determined by automatic biochemical analyzer.

[0111] 2.2 The middle part of the left lobe of the liver was taken for cross-section, frozen section and HE staining. From one end of the liver

[0112] The pathological changes of cells were recorded under a 40x objective lens. The distribution, range and area of lipid droplets in the liver were observed.

[0113] 3. Statistical analysis

[0114] The experimental results were expressed as mean ± standard error. The animal samples belonged to small samples and non-normal distribution, and the significance analysis between the two groups was performed by Mann-Whitney U test, and the statistical software was SPSS 16.0. When P <0.05 was considered to have significant difference.

[0115] 4. Determination results

[0116] As Figure 3 shown, compared with the normal group, the MDA content in the liver tissue of the model group animals was significantly increased, and the difference was significant. Compared with the model group, the high, medium and low dose groups of peony seed oil soft capsules could significantly reduce the MDA content, and the difference was significant (P<0.01). P <0.01).

[0117] As Figure 4 shown, compared with the normal group, the GSH content in the model group was significantly reduced, and the difference was significant. Compared with the model group, the high and medium dose groups of peony seed oil soft capsules could significantly increase the GSH content, and the difference was significant (P<0.01), and the low dose group had no obvious effect. P <0.01).

[0118] As Figure 5 shown, compared with the normal group, the TG content in the liver tissue of the model group animals was significantly increased, and the difference was significant. Compared with the model group, the high, medium and low dose groups of peony seed oil soft capsules could significantly reduce the TG content, and the difference was significant (P<0.01, P <0.05). P

[0119] As Figures 15A-15E shown, the liver tissue structure of the normal group of rats was normal, and there was no obvious lesion in the liver. There were scattered fat droplets in the liver cells. The liver tissue cells of the model group of rats contained a large number of fat droplets, and part of them appeared fusogenic fatty vacuoles. Part of the liver cells were swollen, and there were more inflammatory cell infiltrations. The high, medium and low dose groups of peony seed oil soft capsules could reduce the pathological changes of liver tissue to different degrees, and the high and medium dose groups had better improvement effect on fatty degeneration, and the low dose had relatively weak effect.

[0120] As Figure 6 shown, the pathological score of liver tissue: compared with the normal group, the pathological score of liver tissue of the model group animals was significantly increased, and the difference was significant. Compared with the model group, the high and medium dose groups of peony seed oil soft capsules could significantly reduce the pathological score, and the difference was significant (P<0.01), and the low dose group had no obvious effect. P <0.01).

[0121] ​In addition, the above-mentioned test was also conducted on the samples of Examples 2 and 3, and the results were basically the same as those of Example 1. The above-mentioned results indicate that the peony seed oil soft capsule has obvious protective effect on acute alcoholic liver injury of rats.

[0122] (III) Test of protective effect of the present application on chronic alcoholic liver injury

[0123] 1. Grouping of experimental animals

[0124] 60 SPF SD rats, weighing 180-220 g, half male and half female, were purchased from Pizhou Dongfang Breeding Co., Ltd. They were randomly divided into blank control, model control, high, medium and low groups of peony seed oil soft capsules, 10 animals in each group, half male and half female. The test group was given different concentrations of test substances by gavage at 1 ml / 100 g BW per day, and the model control group and the blank control group were given the same volume of distilled water. The continuous administration time was 14 weeks.

[0125] 2. Experimental procedure

[0126] 50 SPF SD rats, half male and half female, weighing 180-220 g, were randomly divided into blank control group, model control group, high, medium and low dose groups of peony seed oil soft capsules, 10 in each group. At 9 am every day, the three dose groups and the model control group were given corresponding concentrations of liquor by gavage at a volume of 1.1 ml / 100 g·BW (56° Niu Lanchang Erguotou liquor was diluted with distilled water to 11% concentration and started gavage, and the concentration was increased by 3% every day until it was increased to the original concentration, and gavage was performed at 11 ml / kg BW per day, and the continuous gavage time was 12 weeks, and the total modeling time was about 14 weeks). The blank control group was given the same volume of distilled water. At 3 pm every day, the three dose groups were given different concentrations of suspensions by gavage at a volume of 1 ml / 100 g·BW, and the model control group and the blank control group were given the same volume of distilled water, and the continuous gavage time was 14 weeks. The animals in each group were weighed once a week during the test, and the gavage dose of the test substance was adjusted according to the body weight.

[0127] 3. Index determination

[0128] 3.1 After the last gavage of the test solution and the modeling liquor, the arterial blood was taken after 16 hours of fasting, and the supernatant was taken after centrifugation. The content of ALT and AST in serum was determined by biochemical analyzer.

[0129] 3.2 After the blood was taken, all the animals were sacrificed, the complete liver tissue was weighed, and the liver index was calculated.

[0130] 3.3 Part of the liver tissue was taken for pathological examination, the distribution, range and area of lipid droplets in the liver were observed, and the score was given to evaluate the degree of liver tissue fatty degeneration.

[0131] 3.4 A separate quantitative amount of liver tissue was collected to prepare liver homogenate, and malondialdehyde (MDA), reduced glutathione (GSH), triglycerides (TG), and superoxide dismutase (SOD) levels were measured. Differences in liver lipid droplet scores, MDA, GSH, TG, and SOD levels were compared between each dose group and the model control group.

[0132] 4. Statistical Analysis

[0133] Experimental results are expressed as mean ± standard error. The animal samples were small and non-normally distributed; the Mann-Whitney U test was used for significance analysis between the two groups. SPSS 16.0 was used for statistical analysis. P A value <0.05 is considered statistically significant.

[0134] 5. Measurement Results

[0135] like Figures 7-10 As shown, compared with the blank control group, the levels of MDA and TG in the model rats were significantly increased (2.03-fold and 1.43-fold, respectively), while the levels of GSH and SOD were significantly decreased (42.22% and 38.9%, respectively), and the differences were statistically significant. P <0.01); Compared with the model group, the high, medium and low dose groups of peony seed oil soft capsules significantly reduced MDA content (34.05%, 29.73% and 27.57% lower than the model group, respectively), the high and medium dose groups significantly increased GSH content (48.72% and 26.92% higher than the model group, respectively), the high, medium and low dose groups significantly reduced TG content (33.50%, 38.14% and 27.32% lower than the model group, respectively), and the high and medium dose groups significantly increased SOD content (31.74% and 18.56% higher than the model group, respectively).

[0136] like Figures 11-12 As shown, compared with the blank control group, the ALT and AST levels in the model group rats were significantly increased (1.13-fold and 0.94-fold, respectively), indicating significant liver damage. Compared with the model group, the high, medium, and low dose groups of peony seed oil soft capsules significantly reduced ALT levels in rats (37.03%, 38.27%, and 23.46%, respectively), and the high and medium dose groups significantly reduced AST levels (16.00% and 18.67%, respectively), with significant differences. The medium dose showed the most significant effect.

[0137] like Figure 13As shown, compared with the blank control group, the liver index of the model group was significantly increased (by 27.80%), and the difference was statistically significant. Compared with the model group, both the high- and medium-dose groups of peony seed oil soft capsules significantly reduced the liver index of rats (by 14.61% and 17.86% respectively compared with the model group), with the medium-dose group showing a more significant effect and the low-dose group showing no significant effect.

[0138] like Figures 16A-16E As shown, in the control group, the liver lobule structure of rats was normal, the hepatocytes were large, polyhedral, tightly and regularly arranged, without lipid droplet accumulation, and no inflammatory cells were observed. In the model group, the liver lobule structure of rats was incomplete, the hepatocytes were fragmented and loosely arranged, the nuclei were shrunken, and a large number of fat vacuolar lesions were scattered throughout the field of view. Some livers showed punctate necrosis of hepatocytes and inflammatory cell infiltration. In the high- and medium-dose groups of rats treated with peony seed oil soft capsules, the degree of liver tissue damage was reduced, the number and size of fat vacuoles were significantly reduced, the number of fragmented and inflammatory hepatocytes was reduced, and the hepatocyte morphology was relatively regular. The effects of the high- and medium-dose groups were basically similar. In some animals, fat vacuoles essentially disappeared. The low-dose group showed a weaker effect. Figure 14 As shown, the pathological grading results also indicate that compared with the blank control group, the pathological scores of rats in the model group were significantly increased (by 3.4 times), with a statistically significant difference. Compared with the model group, the high, medium, and low dose groups all reduced the pathological scores to varying degrees (by 35.3%, 32.3%, and 17.6% respectively compared with the model group). The high and medium doses had more significant effects and were basically the same, while the low dose had a weaker effect.

[0139] In addition, the samples from Examples 2 and 3 were also subjected to the above tests, and the results were basically the same as those from Example 1. These results suggest that the peony seed oil soft capsules provided by this invention have a significant protective effect against chronic alcoholic liver injury in rats.

[0140] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A composition for protecting against alcoholic liver injury, characterized in that, The raw materials for preparing the composition include the following components in parts by weight: 1-6 parts peony seed oil, 1-8 parts kudzu root, 1-4 parts Japanese raisin tree fruit, and 1-2 parts gardenia. The method for preparing the composition for protecting against alcoholic liver injury includes: S1. Take the seeds of Japanese raisin tree and extract them with water at a ratio of 1:4~6 for 3~5 hours to obtain volatile oil for later use; S2. Mix the residue after extracting the volatile oil with kudzu root and gardenia, add 70-90% v / v ethanol at a ratio of 1:6-10 and extract for 2-3 hours. Collect the extract and recover the ethanol. After concentration, drying and pulverization, obtain the extract powder. S3. Combine the peony seed oil, volatile oil, and extract powder to obtain the final product.

2. The composition for protecting against alcoholic liver injury according to claim 1, characterized in that, The raw materials for preparing the composition include the following components in parts by weight: 3 parts peony seed oil, 4 parts kudzu root, 2 parts Japanese raisin tree fruit, and 1 part gardenia.

3. A method for preparing the composition for protecting against alcoholic liver injury as described in claim 1 or 2, characterized in that, The preparation method includes: S1. Take the seeds of Japanese raisin tree and extract them with water at a ratio of 1:4~6 for 3~5 hours to obtain volatile oil for later use; S2. Mix the residue after extracting the volatile oil with kudzu root and gardenia, add 70-90% v / v ethanol at a ratio of 1:6-10 and extract for 2-3 hours. Collect the extract and recover the ethanol. After concentration, drying and pulverization, obtain the extract powder. S3. Combine the peony seed oil, volatile oil, and extract powder to obtain the final product.

4. The preparation method according to claim 3, characterized in that, In step S1, the material-to-liquid ratio for extraction is 1:5, and the extraction time is 4 hours.

5. The preparation method according to claim 3, characterized in that, In step S2, the material-to-liquid ratio for extraction is 1:8, the ethanol concentration is 80% v / v, and the extraction time is 2.5 h.

6. A peony seed oil soft capsule, characterized in that, The peony seed oil soft capsules consist of contents and capsule material. The raw materials for preparing the contents include the following components in parts by weight: 1-6 parts peony seed oil, 1-8 parts kudzu root, 1-4 parts Japanese raisin tree fruit, 1-2 parts gardenia, and 0.5-1 parts beeswax. The raw materials for preparing the capsule material include the following components in parts by weight: 1-1.1 parts gelatin, 0.4-0.6 parts glycerin, and 1-1.1 parts water. The preparation method of the contents is as follows: a1) Take the seeds of Hovenia dulcis, add water at a ratio of 1:4~6 to extract for 3~5 hours to obtain volatile oil, and set aside for later use; a2) Mix the residue after extracting the volatile oil with kudzu root and gardenia, add 70-90% v / v ethanol at a ratio of 1:6-10 and extract for 2-3 hours. Collect the extract and recover the ethanol. After concentration, drying and pulverization, the extract powder is obtained. a3) Take peony seed oil and beeswax, heat and melt them, let them cool to room temperature, add the extract powder and volatile oil, grind them evenly to obtain the contents.

7. A method for preparing the peony seed oil soft capsules according to claim 6, characterized in that, Includes the following steps: a. Preparation of contents: a1) Take the seeds of Hovenia dulcis, add water at a ratio of 1:4~6 to extract for 3~5 hours to obtain volatile oil, and set aside for later use; a2) Mix the residue after extracting the volatile oil with kudzu root and gardenia, add 70-90% v / v ethanol at a ratio of 1:6-10 and extract for 2-3 hours. Collect the extract and recover the ethanol. After concentration, drying and pulverization, the extract powder is obtained. a3) Take peony seed oil and beeswax, heat to melt, let stand at room temperature, add extract powder and volatile oil, grind evenly to obtain the contents; b. Preparation of capsule material: Mix gelatin, glycerin and water, heat, and cool to form a capsule shell; c. Pressing: After wrapping the contents with rubber, press to obtain the product.

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