CN115501280B - Traditional Chinese medicine composition for improving fatty liver as well as preparation method, extract, application and preparation thereof - Google Patents

Abstract

The invention discloses a traditional Chinese medicine composition for improving fatty liver and its preparation method, extract, application and preparation. The traditional Chinese medicine composition contains the following raw materials: Atractylodes rhizome, tangerine peel, green bark, red peony root, white peony root, Codonopsis root, Guanzhong and Panax notoginseng, the preparation method includes adding formula amounts of Atractylodes rhizome, tangerine peel, green peel, red peony root, white peony root, Codonopsis pilosula, Guanzhong and Panax notoginseng, adding water to extract, and filtrate is combined and concentrated. The extract is obtained from the above-mentioned traditional Chinese medicine composition. The application is the use of traditional Chinese medicine compositions and/or extracts in the preparation of preparations for improving fatty liver. The preparation includes traditional Chinese medicine composition and/or extract. The Chinese medicine composition of the present invention is guided by the theory of "regulating the liver, opening the axis and resolving turbidity", and selects Atractylodes rhizome, tangerine peel, green peel, red peony root, white peony root, Codonopsis root, Guanzhong and Panax notoginseng, and utilizes the ingredients of eight kinds of Chinese medicines. The compound can regulate the liver and spleen, regulate qi and remove blood stasis, and has a significant effect on treating non-alcoholic fatty liver disease and/or hyperlipidemia caused by glucose and lipid metabolism disorders.

Description

Traditional Chinese medicine composition for improving fatty liver and its preparation method, extract, application and preparation

Technical field

The invention belongs to the technical field of traditional Chinese medicine, and specifically relates to a traditional Chinese medicine composition for improving fatty liver and its preparation method, extract, application and preparation.

Background technique

Non-alcoholic fatty liver disease (NAFLD) is a clinical pathological syndrome with diffuse hepatocellular macrovesicular fat as the main pathological feature without a history of excessive drinking. NAFLD is a common clinical disease, and its prevalence is increasing year by year worldwide. The World Health Organization has identified non-alcoholic fatty liver disease as a global public health problem. Therefore, effective prevention and treatment of NAFLD has important practical significance.

There is no specific treatment drug for NAFLD in Western medicine. Clinical treatment mainly focuses on lifestyle intervention, improving insulin resistance, correcting metabolic disorders, and liver-protective treatment. However, the cost of treatment is relatively expensive, and the efficacy, benefits after drug withdrawal, and long-term drug safety are unknown. The sex is not yet clear. Traditional Chinese medicine has unique advantages and good efficacy in preventing and treating NAFLD without obvious adverse reactions, and has broad application prospects. Therefore, finding efficient traditional Chinese medicine compositions from plants to improve fatty liver has become one of the focuses of drug research for the prevention and treatment of NAFLD at home and abroad.

CN104013873A discloses a traditional Chinese medicine composition for treating fatty liver cirrhosis, which is composed of Panax notoginseng, alum, bear bile, turtle shell, turtle shell, raw hawthorn, cooked hawthorn, malt, Shenqu, Guizhi, Lingzhu, Atractylodes, Polyporus, and Poria. , green peel, tangerine peel, fenugreek, Alisma, Cyperus rotunda, salvia, radish seed and licorice, a total of 22 flavors of the formula are combined. The invention regulates the liver and kidneys, strengthens the spleen and removes dampness, removes dampness and removes blood stasis, strengthens the spleen and eliminates stagnation, and soothes the liver. It regulates qi, purges the liver and lowers lipids, removes dampness and reduces swelling, purges and removes accumulation, softens and dissipates stagnation, breaks blood and removes blood stasis, activates blood circulation and unblocks collaterals, reduces swelling and pain, softens and dissipates stagnation, and can be used to treat fatty liver cirrhosis.

CN104042802A discloses a traditional Chinese medicine composition for treating fatty liver. The composition includes the following components: 8-12 parts of Alisma, 5-8 parts of Yincheng, 8-12 parts of tangerine peel, 8-10 parts of Chuanxiong, and Polygonum multiflorum. 3-5 parts, Citrus aurantium 15-20 parts, Hawthorn 15-20 parts, Bupleurum 8-12 parts, Scutellaria baicalensis 12-15 parts, Atractylodes 25-30 parts, White peony root 5-8 parts, Salvia miltiorrhiza 5-8 parts, 3-6 parts of cassia seeds, 5-8 parts of wolfberry, and 8-12 parts of licorice. CN106822781A discloses a traditional Chinese medicine composition for treating fatty liver. The various components of the traditional Chinese medicine composition and their mass proportions are as follows: 80g of Citrus aurantium, 95g of Bupleurum, 200g of hawthorn, 120g of Yinchen, 200g of gardenia, Salvia miltiorrhiza 120g, rhubarb 230g, Alisma 150g, Codonopsis pilosula 200g, Atractylodes 120g, Pinellia 70g, Magnolia officinalis 80g, Puhuang 80g, Coix Seed 350g, White Knot Seed 50g, Talc 230g, Tangerine Peel 95g, Chuanxiong 85g, Turmeric 95g, White Peony Root 200g, cyperus 120g, licorice 115g. The composition of the invention has good therapeutic effect in treating fatty liver.

Glucolipid Metabolic Disorders (GLMD) is a complex disease characterized by disorders of sugar and lipid metabolism, involving genetic, environmental, mental and other factors. Its core mechanisms mainly include: neuroendocrine disorders, insulin resistance, oxidative stress, inflammation and intestinal flora imbalance, etc. The clinical characteristics are mainly single or comorbid diseases such as hyperglycemia, dyslipidemia, non-alcoholic fatty liver disease, overweight, hypertension and atherosclerosis. The applicant's team proposed a comprehensive and integrated treatment strategy of "regulating the liver, opening the axis and resolving turbidity" for the concept of glucose and lipid metabolism diseases. This strategy emphasizes that the liver is the core and hub of the onset of glucose and lipid metabolism diseases, and the prevention and treatment of glucose and lipid metabolism disorders breaks through the current limitations of single disease and single treatment.

Contents of the invention

The purpose of the present invention is to provide a traditional Chinese medicine composition for improving fatty liver and its preparation method, extract, application and medicine under the guidance of the theory of "regulating the liver, opening the axis and resolving turbidity".

In order to achieve the above objects, the present invention adopts the following technical solutions:

A traditional Chinese medicine composition for improving fatty liver, containing the following raw materials: Atractylodes macrocephala, tangerine peel, green peel, red peony root, white peony root, Codonopsis pilosula, Guanzhong and Panax notoginseng.

Preferably, the mass ratio of Qingpi, red peony root, Codonopsis pilosula, Guanzhong and Panax notoginseng is 8-12:9-13:13-17:6-10:8-10; further preferably, it is 10:10:15: 9:9.

More preferably, the traditional Chinese medicine composition for improving fatty liver contains the following raw materials by weight: 8-15 parts by weight of Atractylodes macrocephala, 5-15 parts by tangerine peel, 8-12 parts by Qingpi, 9-13 parts by red peony root, and 8 parts by white peony root. -12 servings, Codonopsis pilosula 13-17 servings, Guanzhong 6-10 servings and Panax notoginseng 8-10 servings.

Most preferably, the traditional Chinese medicine composition for improving fatty liver contains the following raw materials by weight: 12 parts of Atractylodes macrocephala, 10 parts of tangerine peel, 10 parts of green peel, 10 parts of red peony root, 10 parts of white peony root, 15 parts of Codonopsis pilosula, 9 parts of Guanzhong 9 servings and 9 servings of Panax notoginseng.

Preferably, the raw material can be a processed product. Specifically, the Guanzhong can be Atractylodes rhizome or Mianma Guanzhong, the Codonopsis pilosula can be Codonopsis pilosula or cooked Codonopsis pilosula, and the Atractylodes macrocephala can be raw Atractylodes rhizome or bran root. Stir-fried Atractylodes, the tangerine peel can be tangerine peel or steamed tangerine peel.

The invention also provides a traditional Chinese medicine extract for improving fatty liver, which is extracted from the above-mentioned traditional Chinese medicine composition.

Preferably, the extraction is a decoction method, a maceration method, a percolation method, a modified gelatin method, a reflux method, a solvent extraction method, a steam distillation method, a sublimation method, a supercritical fluid extraction method, a membrane separation technology, or an ultramicron method. At least one of crushing technology, traditional Chinese medicine flocculation separation technology, semi-bionic extraction method, ultrasonic extraction method, cyclone extraction method, pressurized countercurrent extraction method, enzymatic method, macroporous resin adsorption method, ultrafiltration method and molecular distillation method.

The invention also provides a preparation method of the above-mentioned traditional Chinese medicine composition for improving fatty liver, which includes the following steps: adding formula amounts of Atractylodes rhizome, Guanzhong, tangerine peel, green peel, red peony root, white peony root, Codonopsis root and Panax notoginseng, adding water to extract 1-3 times , the filtrate is combined and concentrated to obtain the traditional Chinese medicine composition.

Preferably, the extraction is: reflux extraction for 1-2 hours each time.

Preferably, the amount of water added is 8-10 times the total weight of the traditional Chinese medicine composition.

The present invention also provides the use of the above-mentioned traditional Chinese medicine composition in preparing preparations for treating fatty liver.

The present invention also provides the use of the above-mentioned traditional Chinese medicine extract in preparing preparations for treating fatty liver.

The present invention also provides a preparation for treating fatty liver, including the above-mentioned traditional Chinese medicine composition and medically acceptable auxiliary materials.

The present invention also provides a preparation for treating fatty liver, including the above-mentioned traditional Chinese medicine extract and medically acceptable auxiliary materials.

The beneficial effects of the present invention are:

(1) Under the theoretical guidance of the method of "regulating the liver, opening the axis and resolving turbidity", the present invention selects a traditional Chinese medicine for improving fatty liver consisting of atractylodes, tangerine peel, green peel, red peony root, white peony root, Codonopsis root, Guanzhong and Panax notoginseng. The composition (Fat Liver Recipe No. 1), among which, Atractylodes Atractylodes has the functions of strengthening the spleen and removing dampness, green peel soothing the liver and strengthening the spleen, tangerine peel soothing the liver and regulating qi, red peony root activating blood circulation and protecting the liver, white peony root astringing yin and nourishing blood, Codonopsis pilosula tonifying qi and promoting blood circulation, and clearing the body. It protects the liver and reduces inflammation, and Panax notoginseng activates blood circulation and removes blood stasis. The combination of eight kinds of traditional Chinese medicine can regulate the liver and spleen, regulate qi and remove blood stasis. It has a significant effect on treating non-alcoholic fatty liver disease and/or hyperlipidemia caused by glucose and lipid metabolism disorders. .

(2) It was also found that when the mass ratio of Qingpi, red peony root, Codonopsis pilosula, Guanzhong and Panax notoginseng is 8-12:9-13:13-17:6-10:8-10, further synergy can be achieved function to improve the effect of treating fatty liver.

Description of the drawings

Figure 1 shows the liver pathological section results of a mouse model of hyperlipidemia induced by high fat and high sugar.

Figure 2 shows the liver pathological section results of non-alcoholic fatty liver disease (NAFLD) model mice fed high-fat and high-sugar feeding.

Detailed ways

The following describes the embodiments of the present invention through specific examples. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments. Various details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific specific embodiments; it should also be understood that the terms used in the embodiments of the present invention are for describing specific specific embodiments, It is not intended to limit the scope of the present invention.

When the examples give numerical ranges, it should be understood that, unless otherwise stated in the invention, the two endpoints of each numerical range and any value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The present invention does not limit the source of the raw materials used. Unless otherwise specified, the raw materials used in the present invention are all common commercial products in this technical field.

A traditional Chinese medicine composition for improving fatty liver, containing the following raw materials: Atractylodes macrocephala, tangerine peel, green peel, red peony root, white peony root, Codonopsis pilosula, Guanzhong and Panax notoginseng.

Preferably, the mass ratio of Qingpi, red peony root, Codonopsis pilosula, Guanzhong and Panax notoginseng is 8-12:9-13:13-17:6-10:8-10; further preferably, it is 10:10:15: 9:9.

More preferably, the traditional Chinese medicine composition for improving fatty liver contains the following raw materials by weight: 8-15 parts by weight of Atractylodes macrocephala, 5-15 parts by tangerine peel, 8-12 parts by Qingpi, 9-13 parts by red peony root, and 8 parts by white peony root. -12 servings, Codonopsis pilosula 13-17 servings, Guanzhong 6-10 servings and Panax notoginseng 8-10 servings.

Most preferably, the traditional Chinese medicine composition for improving fatty liver contains the following raw materials by weight: 12 parts of Atractylodes macrocephala, 10 parts of tangerine peel, 10 parts of green peel, 10 parts of red peony root, 10 parts of white peony root, 15 parts of Codonopsis pilosula, 9 parts of Guanzhong 9 servings and 9 servings of Panax notoginseng.

Preferably, the raw material can be a processed product. Specifically, the Guanzhong can be Atractylodes rhizome or Mianma Guanzhong, the Codonopsis pilosula can be Codonopsis pilosula or cooked Codonopsis pilosula, and the Atractylodes macrocephala can be raw Atractylodes rhizome or bran root. Stir-fried Atractylodes, the tangerine peel can be tangerine peel or steamed tangerine peel.

The invention also provides a traditional Chinese medicine extract for improving fatty liver, which is extracted from the above-mentioned traditional Chinese medicine composition.

Preferably, the extraction is a decoction method, a maceration method, a percolation method, a modified gelatin method, a reflux method, a solvent extraction method, a steam distillation method, a sublimation method, a supercritical fluid extraction method, a membrane separation technology, or an ultramicron method. At least one of crushing technology, traditional Chinese medicine flocculation separation technology, semi-bionic extraction method, ultrasonic extraction method, cyclone extraction method, pressurized countercurrent extraction method, enzymatic method, macroporous resin adsorption method, ultrafiltration method and molecular distillation method.

The invention also provides a preparation method of the above-mentioned traditional Chinese medicine composition for improving fatty liver, which includes the following steps: adding formula amounts of Atractylodes rhizome, tangerine peel, green peel, red peony root, white peony root, Codonopsis root, Guanzhong and Panax notoginseng, adding water to extract 1-3 times , the filtrate is combined and concentrated to obtain the traditional Chinese medicine composition.

Preferably, the extraction is: reflux extraction for 1-2 hours each time.

Preferably, the amount of water added is 8-10 times the total weight of the traditional Chinese medicine composition.

The present invention also provides the use of the above-mentioned traditional Chinese medicine composition in preparing preparations for treating fatty liver.

The present invention also provides the use of the above-mentioned traditional Chinese medicine extract in preparing preparations for treating fatty liver.

The present invention also provides a preparation for treating fatty liver, including the above-mentioned traditional Chinese medicine composition and medically acceptable auxiliary materials.

The present invention also provides a preparation for treating fatty liver, including the above-mentioned traditional Chinese medicine extract and medically acceptable auxiliary materials.

1. Preventive effect on hyperlipidemia model mice induced by high-fat and high-sugar diet

1Instruments and materials

1.1 Experimental animals

Male KM mice, weighing 18-22 g, were provided by the Guangdong Provincial Medical Experimental Animal Center, license number: SCXK (Guangdong) 2018-0002, experimental unit use license number: SYXK (Guangdong) 2017-0125. The animals were kept in a room temperature of 20-26°C, a relative humidity of 40%-70%, a 12-h light cycle, free access to water and food, and adaptive feeding for 5 days.

1.2 Drugs and reagents

Traditional Chinese medicine composition (Fatty Liver Recipe No. 1); HF60 mouse feed (60kcal% fat calories), U.S. product number 112252, Daitz Biotechnology Co., Ltd.; crystallized fructose, batch number: 20211216C, Shandong Xiwang Sugar Co., Ltd.

1.3 Experimental instruments

TC3KH electronic balance, Changshu Shuangjie Testing Instrument Factory; 5804R low-temperature high-speed centrifuge, Eppendorf, Germany; Scilogex S1010E handheld centrifuge, Shanghai Kehuai Instrument Co., Ltd.; BX53F2 OLYMPUS microscope, Olympus, Japan.

2Experimental methods

2.1 Sample preparation:

Weigh 10 times the amount of the prescribed medicinal materials, add 10 times the amount of pure water, and reflux and extract for 1.5 hours. Continue adding 8 times the amount of pure water to the medicinal residue, and reflux and extract for 1.5 hours. The filtrate is combined and concentrated to a concentration of 0.68g/ml. The liquid is the traditional Chinese medicine composition (Fatty Liver Recipe No. 1).

Specifically, the prescription medicinal materials of Group 1 of Fatty Liver Recipe No. 1 are composed of the following raw materials by weight: 8 parts of Atractylodes macrocephala, 5 parts of tangerine peel (steamed tangerine peel), 12 parts of green peel, 13 parts of red peony root, 8 parts of white peony root, Codonopsis root ( 17 parts of cooked Codonopsis pilosula, 10 parts of Guanzhong (Codonopsis pilosula) and 10 parts of Panax notoginseng;

The prescription medicinal materials of Group 2 of Fatty Liver Recipe No. 1 are composed of the following raw materials by weight: 12 parts of Atractylodes macrocephala, 10 parts of tangerine peel (steamed tangerine peel), 8 parts of green peel, 15 parts of red peony root, 10 parts of white peony root, and dangshen (cooked dangshen). 10 parts, 5 parts of Guanzhong (Wu Mao Fern Guanzhong) and 12 parts of Panax notoginseng;

The prescription medicinal materials of Group 3 of Fatty Liver Recipe No. 1 are composed of the following raw materials by weight: 15 parts of Atractylodes macrocephala, 15 parts of dried tangerine peel (steamed tangerine peel), 8 parts of green peel, 9 parts of red peony root, 12 parts of white peony root, and dangshen (cooked dangshen). 13 parts, 6 parts of Guanzhong (Black-haired Fern Guanzhong) and 8 parts of Panax notoginseng;

The prescription medicinal materials of Group 4 of Fatty Liver Recipe No. 1 are composed of the following raw materials by weight: 12 parts of Atractylodes macrocephala, 10 parts of dried tangerine peel (steamed tangerine peel), 10 parts of green peel, 10 parts of red peony root, 10 parts of white peony root, and dangshen (cooked dangshen). 15 parts, 9 parts of Guanzhong (Black-haired Fern Guanzhong) and 9 parts of Panax notoginseng;

Among them, the mass ratio of Qingpi, red peony root, Codonopsis root, Guanzhong and Panax notoginseng in Group 2 of Fatty Liver Recipe No. 2 is not within the range of 8-12: 9-13: 13-17: 6-10: 8-10 .

2.2 Animal modeling, grouping, and drug administration

Fifty-four male KM mice were adaptively fed for 5 days in the experimental environment. The mice were randomly divided into 6 groups according to the initial TG value, namely: control (CON) group, model (MOD) group, fatty liver recipe No. 1 (E1) group, fatty liver recipe No. 1 2 (E2) group, fatty liver recipe The liver No. 1 prescription 3 (E3) group and the fatty liver No. 1 prescription 4 (E4) group were administered continuously for 4 weeks by gavage. During this period, they were fed high-fat feed and sugar water (fructose concentration was 20%). After the experiment is completed, material collection and related testing are carried out.

2.3 Indicator detection

2.3.1 Determination of serum and liver index

After 4 weeks of administration, the mice were fasted for 12 hours in advance (water was not allowed), the animals were anesthetized, the eyeballs were removed and blood was collected in a 1.5 mL EP tube, left to stand for 2 hours, centrifuged at 3500 r/min in a pre-cooled centrifuge at 4°C for 10 minutes, and then the blood was collected. Serum serum was used to determine TG levels. The liver was weighed and the liver index calculated.

2.3.2 Observation of liver tissue pathological sections

The same part of the liver tissue of each mouse was fixed in 4% paraformaldehyde, followed by dehydration, embedding and sectioning, and HE staining was performed to observe the pathological changes of the liver.

Preparation of paraffin sections: Fresh liver tissue is fixed in fixative for more than 24 hours, and the procedures are performed sequentially. Remove the tissue from the fixative, use a scalpel to smooth the target tissue in a fume hood and place it in a dehydration box. Put the dehydration box into the dehydrator and perform dehydration and wax impregnation through gradient alcohol in sequence. The wax-impregnated tissue is embedded and trimmed in an embedding machine. Cool the trimmed wax block at -20°C, and then place the cooled wax block into paraffin microtome slices with a thickness of approximately 4 μm. The slices were floated on warm water at 40°C in a spreading machine to flatten the tissue. The tissue was picked up by a glass slide and baked in a 60°C oven. After the water-baked dry wax is baked, take it out and store it at room temperature for later use.

HE staining: Place the sections in xylene for 30 min - absolute ethanol for 5 min - 95% ethanol for 5 min - 85% ethanol for 5 min - 75% ethanol - dd water for 2 min. Stain the sections in hematoxylin staining solution for 2 minutes, wash with tap water, differentiate with differentiation solution, wash with tap water, stain in eosin staining solution for 5 minutes, and rinse with running water. The slices were dehydrated in gradient ethanol of 70%, 80%, 90%, and 100% for 10 s each, and then dried. The sections were placed in xylene for 10 minutes to make them transparent, dried, and sealed with neutral gum.

2.4 Statistical methods

The experimental data were processed using IBM SPSS Statistics, and the measurement data were processed using means; independent sample t test was used for comparison between two groups, and ANOVA analysis of variance was used for comparison between multiple groups. P<0.05 means statistically significant.

3Experimental results

3.1 Effect of Fatty Liver No. 1 Recipe on Serum TG Levels in NAFLD Model Mice

Before the experiment, TG was detected in the mice, and there was no significant difference between each group (P>0.05). After 4 weeks of administration (28 days after administration), compared with the CON group, TG in the MOD group was significantly increased (P<0.05); compared with the MOD group, TG in the E1 and E3 groups was significantly decreased (P<0.05), and in the E4 group TG was extremely significantly reduced (P<0.01). The results are shown in Table 1.

Table 1 Effect of Fatty Liver Recipe No. 1 on serum TG levels in NAFLD model mice (mmol/L, )

Note: ^* P<0.05, ^** P<0.01, compared with CON group; ^# P<0.05, ^## P<0.01, compared with MOD group.

3.2 Effect of Fatty Liver Formula No. 1 on liver index of NAFLD model mice:

As can be seen from Table 2, there was no significant difference in the liver index of mice in each group after administration (P>0.05).

Table 2 Effect of Fatty Liver No. 1 Recipe on Liver Index of NAFLD Model Mice

Note: ^* P<0.05, ^** P<0.01, compared with CON group; ^# P<0.05, ^## P<0.01, compared with MOD group.

3.3 Effect of Fatty Liver Formula No. 1 on liver morphology in NAFLD model mice

HE staining results (Figure 1) showed that the liver lobule structure of the liver tissue of mice in the CON group was normal, the liver sinusoids were clear, the liver cords were neatly arranged, and the hepatocyte morphology was normal. After being fed high-fat feed and sugar water (20% fructose) for 4 weeks, fat vacuoles of different sizes were visible in the liver tissue of mice in the MOD group, and the structure of the liver lobules was blurred. Compared with the MOD group, the number of fat vacuoles in the E1 group was significantly reduced, and the cells were tightly arranged. The fat vacuoles in the E2 group were obvious, and the liver cells in the visual fields of the E3 and E4 groups were neatly arranged and in normal shape, and the drug administration effect was obvious.

To sum up, after KM mice were fed high-fat feed and sugar water (20% fructose) for 4 weeks (i.e. 28 days), the blood lipid TG level increased significantly (P<0.05), and liver cells showed fatty degeneration: lipid droplets stacked, Liver cells are not tightly packed. At the same time, Fatty Liver Recipe No. 1 was administered intragastrically. Based on the results of TG and liver tissue pathological sections, it can be found that the Fatty Liver Recipe No. 1 of the present invention significantly reduced the TG level of mice (P<0.05), and the liver cells were improved. Improvement, indicating that Fatty Liver Recipe No. 1 has the effects of regulating blood lipids and protecting the liver. No damage to other organs was found, and it is highly safe.

2. Study on the prevention and treatment of fatty liver formula No. 1 in mice model of non-alcoholic fatty liver disease (NAFLD) caused by high-fat and high-sugar feeding

1Instruments and materials

1.1 Experimental animals

Fifty-two male KM mice, weighing 18-22 g, were provided by the Guangdong Provincial Medical Experimental Animal Center, license number: SCXK (Guangdong) 2018-0002, and experimental unit use license number: SYXK (Guangdong) 2017-0125. The animals were kept in a room temperature of 20-26°C, a relative humidity of 40%-70%, a 12-h light cycle, free access to water and food, and adaptive feeding for 5 days.

1.2 Drugs and reagents

Influenced by the same 1.2 drugs and reagents in "1. Preventive effect on hyperlipidemia model mice induced by high-fat and high-sugar".

1.3 Experimental instruments

Same as 1.3 experimental equipment in "1. Preventive effect on hyperlipidemia model mice induced by high-fat and high-sugar".

2Experimental methods

2.1 Sample preparation:

Same as 2.1 sample preparation in "1. Preventive effect on hyperlipidemia model mice induced by high fat and high sugar".

2.2 Animal modeling, grouping, and drug administration

Fifty-two male KM mice were adaptively fed for 1 week in the experimental environment. One week later, 8 mice were selected as the blank (CON) group and fed with ordinary feed and drinking water, while the remaining mice were fed with high-fat feed and fructose solution (20%). After 2 weeks, the high-fat and high-sugar group was randomly divided into 5 groups according to TG value, namely: model (MOD) group, fatty liver recipe No. 1 (E1) group, fatty liver recipe No. 1 2 (E2) group, fatty liver I recipe Group No. 3 (E3) and No. 4 (E4) Group No. 1 Prescription for Fatty Liver Disease were administered continuously for 10 weeks by intragastric administration. During the period, the CON group was fed ordinary feed and pure water, while the model and drug treatment groups were fed high-fat feed and sugar water. After the experiment is completed, material collection and related testing are carried out.

2.3 Indicator detection

2.3.1 Determination of serum and liver biochemical indicators

After 10 weeks of administration, the mice were fasted 12 hours in advance (water was not allowed), the animals were anesthetized, the eyeballs were removed and blood was collected in a 1.5 mL EP tube, left to stand for 2 hours, centrifuged at 3500 r/min in a 4°C pre-cooled centrifuge for 10 minutes, and the blood was collected. Serum serum was used to determine the concentrations of ALT and AST. Prepare liver tissue homogenate and detect the concentration of TG in liver tissue.

2.3.2 Pathological examination of liver tissue

The same part of the liver tissue of each mouse was fixed in 4% paraformaldehyde, followed by dehydration, embedding, and sectioning, and HE staining (specifically as follows) was performed to observe the pathological changes in the liver. The NAFLD activity score was calculated according to Table 3 below. Statistical evaluation.

Table 3 Liver tissue NAS score table

Preparation of paraffin sections: Fresh liver tissue is fixed in fixative for more than 24 hours, and the procedures are performed sequentially. Remove the tissue from the fixative, use a scalpel to smooth the target tissue in a fume hood and place it in a dehydration box. Put the dehydration box into the dehydrator and perform dehydration and wax impregnation through gradient alcohol in sequence. The wax-impregnated tissue is embedded and trimmed in an embedding machine. Cool the trimmed wax block at -20°C, and then place the cooled wax block into paraffin microtome slices with a thickness of approximately 4 μm. The slices were floated on warm water at 40°C in a spreading machine to flatten the tissue. The tissue was picked up by a glass slide and baked in a 60°C oven. After the water-baked dry wax is baked, take it out and store it at room temperature for later use.

HE staining: Place the sections in xylene for 30 min - absolute ethanol for 5 min - 95% ethanol for 5 min - 85% ethanol for 5 min - 75% ethanol - dd water for 2 min. Stain the sections in hematoxylin staining solution for 2 minutes, wash with tap water, differentiate with differentiation solution, wash with tap water, stain in eosin staining solution for 5 minutes, and rinse with running water. The slices were dehydrated in gradient ethanol of 70%, 80%, 90%, and 100% for 10 s each, and then dried. The sections were placed in xylene for 10 minutes to make them transparent, dried, and sealed with neutral gum.

2.4 Statistical methods

The experimental data were processed using IBM SPSS Statistics, and the measurement data were processed using means; independent sample t test was used for comparison between two groups, and ANOVA analysis of variance was used for comparison between multiple groups. P<0.05 means statistically significant.

3Experimental results

3.1 Effect of Fatty Liver Formula No. 1 on serum ALT and AST levels in NAFLD model mice

After the experiment, the mouse serum ALT and AST were detected. Compared with the CON group, there were significant differences in ALT and AST in mice in the MOD group (P<0.05); compared with the MOD group, ALT in each administration group was significantly reduced, with a significant difference (P<0.05). The results are shown in the table 4.

Table 4 Effects of Fatty Liver No. 1 recipe on serum ALT and AST levels in NAFLD model mice (U/L, )

Note: ^* P<0.05, ^** P<0.01, compared with CON group; ^# P<0.05, ^## P<0.01, compared with MOD group.

3.2 Effect of Fatty Liver No. 1 Recipe on liver TG levels in NAFLD model mice

After the experiment, the liver tissue was weighed and homogenized. After centrifugation, the supernatant was taken to detect TG. Compared with the CON group, the TG of mice in the MOD group increased, and there was a significant difference (P<0.01); compared with the MOD group, the TG of each administration group tended to decrease, but there was no significant difference (P>0.05) , the results are shown in Table 5.

Table 5 Effect of Fatty Liver No. 1 Recipe on liver TG levels in NAFLD model mice (mmol/L, )

Note: ^* P<0.05, ^** P<0.01, compared with CON group; ^# P<0.05, ^## P<0.01, compared with MOD group.

3.3 Effects of Fatty Liver Recipe No. 1 on pathological changes in liver tissue of NAFLD model mice

The results of HE staining (see Figure 2) showed that the liver lobules of the mice in the CON group had normal structure, the liver cells were neatly arranged, normal in shape, and the central vein was obvious; in the MOD group, obvious lesions appeared in the field of view, and the liver cells centered on the central vein appeared. Fatty degeneration, accompanied by lipid droplets of varying sizes, and small focal inflammatory cell infiltrates around the veins. Compared with the MOD group, the lipid droplets in the E2 group were obvious and the area was reduced. The effect of administration of E1, E3, and E4 groups was obvious, the number and size of lipid droplets were significantly reduced, and the hepatocyte structure was clear and tightly arranged. The results of the effect of Fatty Liver Recipe No. 1 on the pathological scores of liver tissue are shown in Table 6:

Table 6 Effects of Fatty Liver Recipe No. 1 on liver tissue pathology scores

Note: ^* P<0.05, ^** P<0.01, compared with CON group; ^# P<0.05, ^## P<0.01, compared with MOD group.

In summary, KM mice were continuously fed with the Chinese medicinal composition of the present invention for 10 weeks while maintaining high-fat feed and sugar water (20% fructose). Pathological section results showed that the liver cells of mice in the MOD group had undergone significant degeneration: a large number of fat vacuoles appeared, and the morphology of the liver lobules changed significantly. The pathological sections of E1, E3 and E4 of the tested Chinese medicinal composition (Fatty Liver No. 1 prescription) group showed that the liver tissue structure was significantly improved, indicating that the Chinese medicinal composition of the present invention (Fatty Liver No. 1 prescription) has hepatoprotective effects on NAFLD model mice. It protects the liver, has no damage to other organs, and is highly safe.

The above is a further description of the present invention with reference to specific embodiments, but these embodiments are only exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solution of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and substitutions all fall within the protection scope of the present invention.

Claims (6)

1. A traditional Chinese medicine composition for improving fatty liver, characterized in that it is composed of the following raw materials by weight: 8-15 parts by weight of Atractylodes macrocephala, 5-15 parts by tangerine peel, 8-12 parts by Qingpi, 9-13 parts by red peony root, Atractylodes macrocephala by weight. 8-12 parts of peony root, 13-17 parts of Codonopsis pilosula, 6-10 parts of Guanzhong and 8-10 parts of Panax notoginseng, and the mass ratio of Qingpi, red peony root, Codonopsis pilosula, Guanzhong and Panax notoginseng is 8-12:9 -13:13-17:6-10:8-10. 2. The traditional Chinese medicine composition according to claim 1, characterized in that it is composed of the following raw materials by weight: 12 parts of Atractylodes macrocephala, 10 parts of tangerine peel, 10 parts of green peel, 10 parts of red peony root, 10 parts of white peony root, and 15 parts of Codonopsis pilosula. , Guanzhong 9 copies and Panax notoginseng 9 copies. 3. The traditional Chinese medicine composition according to claim 1, characterized in that the Guanzhong is Atractylodes rhizome or Mianma Guanzhong, the Codonopsis pilosula is Codonopsis pilosula or cooked Codonopsis pilosula, and the Atractylodes rhizome is raw Atractylodes rhizome or bran. Fried Atractylodes, the tangerine peel is tangerine peel or steamed tangerine peel. 4. The preparation method of the traditional Chinese medicine composition according to any one of claims 1 to 3, characterized in that it includes the following steps: adding the formula amounts of Atractylodes Rhizome, dried tangerine peel, green bark, red peony root, white peony root, Codonopsis pilosula, Guanzhong and San 7. Add water and extract 1-3 times, combine the filtrate and concentrate to obtain the traditional Chinese medicine composition. 5. Application of the Chinese medicinal composition according to any one of claims 1 to 3 or the Chinese medicinal composition prepared by the preparation method according to claim 4 in the preparation of preparations for treating fatty liver. 6. A preparation for treating fatty liver, characterized by comprising the Chinese medicinal composition according to any one of claims 1 to 3 or the Chinese medicinal composition prepared by the preparation method according to claim 4 and medically acceptable auxiliary materials.