CN107029074A - A kind of pharmaceutical composition and preparation method and purposes for treating fatty liver - Google Patents

Abstract

The invention discloses a pharmaceutical composition for treating fatty liver, which is a preparation prepared from the following raw materials in proportion by weight: 10-12.5 parts of Epimedium, 15-20 parts of Radix Astragali, 5-7.5 parts of Poria cocos, Atractylodes 5‑7.5 parts, Alisma 7.5‑10 parts, tangerine peel 7.5‑10 parts, pinellia 7.5‑10 parts, raw hawthorn 15‑20 parts, and salvia miltiorrhiza 15‑20 parts. The invention also provides the preparation method and application of the pharmaceutical composition. The pharmaceutical composition of the invention has precise compatibility, and the flavors of the medicines complement each other, has remarkable curative effect for treating fatty liver, and provides a new choice for clinical treatment.

Description

A kind of pharmaceutical composition for treating fatty liver and its preparation method and application

technical field

The invention relates to a pharmaceutical composition for treating fatty liver, a preparation method and application thereof, and belongs to the field of traditional Chinese medicines.

Background technique

Nonalcoholic fatty liver disease (NAFLD) is a metabolic stress-induced liver injury closely related to insulin resistance (insulin resistance, IR) and genetic susceptibility, and its pathological changes are similar to those of alcoholic liver disease (Alcoholic liver disease). Fatty liver disease (ALD) is similar, but the patient has no history of excessive drinking. The disease spectrum includes nonalcoholic fatty liver (NAFL), nonalcoholic steatohepatitis (NASH) and its related cirrhosis and Hepatocellular Carcinoma (HCC). With the improvement of living standards and changes in lifestyle and dietary structure, the incidence of NAFLD has been increasing, and it has gradually become one of the most important public health problems in the world.

Survey data show that NAFLD is the most common cause of chronic liver disease in western countries, with an overall prevalence rate of about 25%, and the proportion increases to 70% among obese people and type 2 diabetes patients. The epidemiological survey in our country is not optimistic. The incidence of NAFLD is as high as 20.7%. At present, in the prevalence of obesity and metabolic syndrome in China, NAFLD has replaced chronic hepatitis B as the number one chronic liver disease. As far as the harm of NAFLD is concerned, it is similar to alcoholic liver disease and viral liver disease. Through liver inflammation, liver cirrhosis, liver cancer, and even liver failure can occur. But relatively speaking, the probability of liver cirrhosis and hepatocellular carcinoma in NAFLD is relatively low; in addition, the process from steatohepatitis to liver cirrhosis and liver cancer takes longer for NAFLD than viral hepatitis. However, in developed countries in Europe and the United States, especially in the United States, the liver transplantation caused by NAFLD has ranked second, second only to chronic hepatitis C; among the causes of HCC, the growth rate of NAFLD has risen rapidly, and it seems to have replaced Alcoholic liver disease is the second leading cause. Therefore, research on the pathogenesis and drug treatment of NAFLD is of great significance.

Currently, there are no specific means and drugs for the treatment of NAFLD. Treatment mainly includes basic treatment and drug treatment. The basic treatment is to improve lifestyle, such as controlling diet, strengthening exercise and reducing weight. Drug therapy includes five categories: (1) Drugs for improving insulin resistance: commonly used drugs are insulin sensitizers, including biguanides represented by metformin and thiazolidinediones represented by rosiglitazone and pioglitazone; 2) Lipid-lowering drugs: represented by fibrates and statins, statins are mainly used to lower TC and LDL-C, and fibrates are mainly used for hyperlipidemia mainly caused by elevated TG; Liver anti-inflammatory drug: Licorice preparation exerts its strong hepatoprotective and anti-inflammatory effect through its steroid effect. Polyene phosphatidylcholine capsules can stabilize, protect and repair liver cell membrane through specific combination with liver cell membrane, and promote liver cell regeneration. Such as ursodeoxycholic acid, which works by preventing apoptosis and inhibiting inflammatory response; (4) antioxidants: such as vitamin E, which can delay the progression of NAFLD to The process of NASH; (5) Probiotics: Overgrowth of intestinal bacteria can produce endogenous alcohol, release endotoxin, stimulate the production of inflammatory cytokines and participate in the damage of NAFLD. However, these drugs have certain side effects: such as thiazolidinediones and lipid-lowering drugs have certain liver damage effects, and lipid-lowering drugs can rebound after drug withdrawal, and cannot effectively improve lipid deposition in the liver; biguanides can cause lactic acid Risk of acidosis etc. Therefore, there is currently no drug approved for the treatment of NAFLD.

Relying solely on liver-protecting anti-inflammatory drugs, lipid-lowering drugs, and insulin resistance-improving drugs is not ideal for the treatment of NAFLD, and the curative effect is single, with different toxicity and adverse reactions. Compared with western medicine, traditional Chinese medicine has the characteristics of multi-channel, multi-angle, multi-level, and multi-target comprehensive pharmacological effects. Traditional Chinese medicine has obvious advantages in the treatment of NAFLD, mainly because traditional Chinese medicine is convenient for specifying individualized treatment plans. Compared with simple lipid-lowering, Liver-protective regimens are more effective. There are many types of traditional Chinese medicine compound prescriptions or unit traditional Chinese medicines that can be used to treat NAFLD, with high safety, stable and long-lasting curative effect in the prevention and treatment of NAFLD, less toxic and side effects, and low price.

At present, there are many reports on traditional Chinese medicine for the treatment of fatty liver, such as: Zhong Yanchun, Progress in the treatment of fatty liver with traditional Chinese medicine, Journal of Liaoning University of Traditional Chinese Medicine, February 2008, Volume 10, No. 2, according to the etiology, pathogenesis and dialectical analysis of fatty liver Many different prescriptions have been disclosed for the treatment of this disease. At present, there are still many problems in the treatment of this disease with traditional Chinese medicine, such as TCM diagnosis, syndrome differentiation, judgment standards and curative effect judgment standards are not uniform, objective indicators are not clear, and long-term curative effect observations are few. There is a lack of large-sample, multi-center, strictly randomized controlled studies, and there are very few studies on the treatment mechanism and pharmacological effects. It is necessary to continuously improve and determine the unified syndrome classification, diagnostic criteria, curative effect judgment criteria and quantitative objective indicators for fatty liver treatment. Extensive and scientific research on the mechanism of traditional Chinese medicine has provided reliable and effective evidence for clinical treatment of this disease. Patent application number: CN201310305337.9, Invention name: A traditional Chinese medicine composition for treating fatty liver and its preparation method, the invention discloses a traditional Chinese medicine composition for treating fatty liver and its preparation method. Made by weight ratio: malt, Poria cocos, Atractylodes macrocephala, Polygonum multiflorum, Alisma, Polygonatum, Astragalus, Salvia miltiorrhiza, Polygonum cuspidatum, lotus leaf, red peony, herb, parasitic, Morinda officinalis, Toosendan, Angelica, Bupleurum, Cassia, Fa Pinellia, Coptidis Rhizome, Cortex Moutan, Green Peel, Citrus Citrifolia, Codonopsis, Tangerine Peel, Curcuma, Licorice, Sanleng, Curcuma. The amount of prescription raw materials is large, which is not conducive to clinical use and industrialized production.

Contents of the invention

The object of the present invention is to provide a new traditional Chinese medicine composition for treating fatty liver. Another object of the present invention is to provide the preparation method and application of the pharmaceutical composition.

The pharmaceutical composition for treating fatty liver of the present invention is a preparation prepared from the following raw materials in weight ratio:

Epimedium 10-12.5 parts, Astragalus 15-20 parts, Poria cocos 5-7.5 parts, Atractylodes macrocephala 5-7.5 parts, Alisma 7.5-10 parts, tangerine peel 7.5-10 parts, pinellia 7.5-10 parts, raw hawthorn 15- 20 parts, 15-20 parts of Danshen.

Preferably, it is a preparation prepared from raw materials in the following weight ratio:

10 parts of Epimedium, 15 parts of Astragalus, 7.5 parts of Poria cocos, 7.5 parts of Atractylodes macrocephala, 10 parts of Alisma, 10 parts of tangerine peel, 7.5 parts of pinellia, 15 parts of raw hawthorn, and 15 parts of Danshen.

Wherein, the pharmaceutical composition is a preparation prepared from raw drug powder or water or organic solvent extract of the crude drug as the active ingredient, plus pharmaceutically acceptable excipients or auxiliary ingredients.

Preferably, the formulation is an oral formulation. Further preferably, the oral preparations are granules, tablets, capsules, or oral liquids.

The present invention also provides a preparation method of the aforementioned pharmaceutical composition, which comprises the following steps:

(1) Take the raw materials of each weight proportion;

(2) A preparation prepared by powdering, or extracting by adding water or an organic solvent, and adding pharmaceutically acceptable excipients or auxiliary ingredients.

The present invention also provides the application of the aforementioned pharmaceutical composition in the preparation of medicine for treating fatty liver. Wherein, the medicine is a medicine for treating non-alcoholic fatty liver.

The present invention also provides the use of the pharmaceutical composition in the preparation of medicines for preventing liver fibrosis.

The present invention also provides the application of the aforementioned pharmaceutical composition in the preparation of medicines with the functions of warming yang, invigorating spleen, eliminating dampness and resolving phlegm.

Non-alcoholic fatty liver of obesity, yang deficiency and phlegm-dampness blockage is a common clinical syndrome, with a large number of patients and a large base of disease sources, which seriously threatens the health of the people. There is still a lack of reliable drugs for this syndrome in Chinese medicine. According to the etiology and pathogenesis of this syndrome type, the pathogenesis theory of "spleen deficiency and stagnation of dampness, combined phlegm and blood stasis" is put forward, and the basic treatment principle is "warming yang and strengthening the spleen, eliminating phlegm and dispelling blood stasis". In the prescription of the pharmaceutical raw material of the present invention, astragalus tonifies the spleen and helps the spleen to promote clear yang; Harmonizes the middle and eliminates stagnation; Poria cocos is sweet and bland, invigorates the spleen, invigorates the middle, diures water and eliminates dampness, two medicines are used together, one strengthens and the other penetrates, and water and dampness have a way out, so the spleen can be strengthened and damp can be eliminated; Spleen invigorating; Alisma diuretic and diuresis; tangerine peel, pinellia, tangerine peel to regulate qi and invigorate the spleen, harmonize the stomach and resolve phlegm, pinellia to dry dampness and resolve phlegm, eliminate swelling and stagnation, strengthen the spleen and stop vomiting, both enter the spleen meridian, two medicines Participating and promoting each other, so the spleen can be strengthened, dampness can be removed, phlegm can disappear, and the movement of qi is unobstructed; raw hawthorn is sour and sweet in nature, good at eliminating stagnation and stagnation, and is mainly used for meat stagnation, good at promoting qi and dissipating blood stasis to relieve pain; stasis. The first important thing is to invigorate Qi and invigorate the spleen, warm the spleen yang, and remove phlegm and blood stasis.

The pharmaceutical composition of the invention has precise compatibility, and the flavors of the medicines complement each other, has clear curative effect on fatty liver and has little toxic and side effects, and provides a new choice for clinical treatment of fatty liver.

Apparently, according to the above content of the present invention, according to common technical knowledge and conventional means in this field, without departing from the above basic technical idea of the present invention, other various forms of modification, replacement or change can also be made.

The above-mentioned content of the present invention will be further described in detail below through specific implementation in the form of examples. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Description of drawings

Figure 1. Results of Oil Red O staining. A. Chinese medicine high-dose group, B. Middle-dose group, C. Low-dose group, D. Positive drug group, E. Model group, F. Blank group

Figure 2. qPCR results. A. TLR4, B. NF-κB, C. TGF-β1, D. Smad3

detailed description

Embodiment 1: the preparation of medicine of the present invention

Take 10g of Epimedium, 15g of Astragalus, 7.5g of Poria, 7.5g of Atractylodes Rhizoma, 10g of Alisma, 10g of Tangerine Peel, 7.5g of Pinellia, 15g of Raw Hawthorn, 15g of Salvia, add 10 times the amount of water, soak overnight, make up the water absorption, and fry. Boil 3 times, each time for 1 hour, filter, and combine the filtrate, that is.

Usage and dosage: Oral, one dose a day, 3 times a day.

Embodiment 2: the preparation of pharmaceutical granule of the present invention

Take 10g of Epimedium, 15g of Astragalus, 7.5g of Poria, 7.5g of Atractylodes Rhizoma, 10g of Alisma, 10g of Tangerine Peel, 7.5g of Pinellia, 15g of Raw Hawthorn, 15g of Salvia, add 10 times the amount of water, soak overnight, make up the water absorption, and fry. Boil 3 times, each time for 1 hour, filter, combine the filtrate, and concentrate the filtrate under reduced pressure to form an extract with a relative density of 1.25 (60°C); mix the extract with 3 times the amount of dextrin to prepare a soft material, and granulate with a swing Mechanically granulate, dry at 60°C for 0.5h, granulate, sieve out fine particles, pack, and get ready.

Embodiment 3: the preparation of pharmaceutical tablet of the present invention

Take 10g of Epimedium, 15g of Astragalus, 7.5g of Poria, 7.5g of Atractylodes Rhizoma, 10g of Alisma, 10g of Tangerine Peel, 7.5g of Pinellia, 15g of Raw Hawthorn, 15g of Salvia, add 10 times the amount of water, soak overnight, make up the water absorption, and fry. Boil 3 times, each time for 1 hour, filter, combine the filtrate, and concentrate the filtrate under reduced pressure to form an extract with a relative density of 1.25 (60°C); mix the extract with 3 times the amount of dextrin to prepare a soft material, and granulate with a swing Mechanically granulate, dry at 60°C for 0.5h, and compress with a single-punch tablet machine to prepare tablets with a size of 0.3g.

The beneficial effects of the present invention are demonstrated through specific experiments below.

Test Example 1 Study on Cellular Pharmacodynamics and Mechanism of Drug-containing Serum of the Present Invention on Nonalcoholic Fatty Liver Disease

1. Experimental materials

1.1 Instruments and reagents

CO2 incubator (Nuare, USA), ultra-clean bench (Jinan Zhuoao), inverted microscope (Olympus, Japan), desktop low-speed centrifuge (Thermo, USA), ultrasonic cell disruptor (Nanjing Xingchen Technology Co., Ltd.), enzyme Standard instrument (Thermo Company, USA), Thermo Company (Zhejiang Saide), ABI 7500 real-time fluorescent quantitative PCR instrument (ABI7500).

DMEM medium (Gibco, USA), fetal bovine serum (Hangzhou Sijiqing Bioengineering Materials Co., Ltd.), trypsin (Gibco, USA), oleic acid (MP, USA), oil red O (SIGMA, USA), triglyceride Ester (TG) Determination Kit (Nanjing Jiancheng Biotechnology Co., Ltd.), M-MLV Frist Stand cDNA Synthesis Kit (OMEGA Company), SYBR Green PCR Master Mix Kit (Applied biosystems Company), Tirzol (AMBION Company), liver cancer cells Strain HepG2 (derived from the cell bank of the Central Laboratory of the Affiliated Hospital of Chengdu University of Traditional Chinese Medicine). Polyene phosphatidylcholine capsules (228mg*24 capsules, Sanofi Pharmaceutical Co., Ltd.), purchased from the Affiliated Hospital of Chengdu University of Traditional Chinese Medicine.

1.2 Experimental animals

Seventy clean-grade male Wistar rats, weighing 200±20g, were provided by Sichuan Jianyang Dashuo Biological Company, and the animal qualification certificate number: SCXK (Sichuan) 2008-011.

2. Experimental method

2.1 Preparation of the drug-containing serum of the present invention

70 male Wistar rats were randomly divided into 5 groups, namely high dose of traditional Chinese medicine, medium dose of traditional Chinese medicine, low dose of traditional Chinese medicine, positive control group (polyene phosphatidylcholine capsule group), and blank group, with 14 rats in each group. According to the body surface area conversion algorithm of people and rats, give the medicine of the present invention (prepared in embodiment 1) high, middle and low dosage groups to gavage respectively 18.8g/kg, 9.4g/kg, 4.7g/kg, positive control group 122.14mg /kg, the daily dose was administered twice by stomach with an interval of 8 hours, and the blank group was given an equal volume of distilled water for 7 consecutive days. One hour after the last administration, blood was collected from the abdominal aorta of the rats, left to stand, and centrifuged to obtain drug-containing serum.

2.2 HepG2 cell culture and grouping

HepG2 cells were inoculated at 1×10 ⁵ cells/well in a 12-well culture plate to test the cells in the logarithmic growth phase. When the cells grow to 70-80%, the original culture medium is discarded, and an appropriate amount of PBS buffer is added to wash twice.

The experiment is divided into 6 groups:

Blank group: adding ordinary medium for culture;

Model group: add medium containing 0.25mM oleic acid.

Positive drug group: add 10% positive drug-containing serum to the culture medium containing 0.25mM oleic acid.

High, medium and low dose groups of traditional Chinese medicine: Add 10% serum containing high, medium and low doses of traditional Chinese medicine to the culture solution containing 0.25mM oleic acid.

2.3 Oil red O staining

The cells were cultured according to the experimental group for 24 hours, the medium was discarded, washed 3 times with PBS, fixed with 4% paraformaldehyde for 30 minutes, washed 2 times with PBS, added 60% isopropanol synchronization solution, and incubated in the dark with oil red O working solution 30min, 60% isopropanol for color separation for about 30s until the background color disappears, wash with ddH ₂ O for 3 minutes, counterstain with hematoxylin for 3-5 minutes, wash 3 times with ddH ₂ O for 2 minutes each time, seal with ddH ₂ O, and place it upside down Cell lipid droplets were observed under a microscope, and photographs were taken with different magnifications and different fields of view.

2.4 Determination of TG/protein content in cells

Treat the cells according to the experimental grouping, discard the medium, wash with PBS 3 times, add 200 μL of 0.25% trypsin to digest, absorb the trypsin, add an appropriate amount of DMEM containing 10% FBS to resuspend the cells, centrifuge, and add 1ml of the cells to lyse The solution was crushed under an ultrasonic cell disruptor, incubated on ice for 20 min, and centrifuged to obtain the supernatant. The intracellular protein content was determined according to the instructions of the BCA protein quantification kit, and the intracellular TG content was determined according to the instructions of the triglyceride kit.

Calculated according to the formula: intracellular triglyceride content (mmol/gprot) = (sample OD value - blank OD value) / (standard OD value - blank OD value) × calibrator concentration (2.26mmol/L) ÷ test sample protein Concentration (gprot/L)

2.5 qPCR method to detect the expression of target genes (TLR4, NF-κB, TGF-β1 and Smad3) at the mRNA level in HepG2 cells

Collect the cell culture fluid of each group in the well plate, collect the supernatant after centrifugation, use trizol reagent to extract RNA, and detect TLR4, NF-κB, TGF- The expression levels of β1 and Smad3 at the mRNA level.

3. Statistical processing

SPSS 21.0 software was used to process the data, and the experimental results were expressed as mean ± standard deviation Indicates that the data conforms to normal distribution and homogeneity of variance, so independent samples t-test is used to compare the mean of blank group and model group, and p<0.05 indicates statistical significance.

4. Experimental results

4.1 Oil red O staining results of HepG2 cells after 24 hours of drug intervention in each group

In the blank group, the cell edges were clear, the cytoplasm was abundant, the nuclear membrane was complete, the nuclei were stained blue, and no obvious red lipid droplets were seen in the cells; many red lipid droplets of different sizes appeared in the cells of the model group, and some of the cells with severe fat change even fused Into a piece; in the positive drug group, the high-dose, medium-dose, and low-dose groups of traditional Chinese medicine, the nuclei were stained blue, and red lipid droplets appeared in different degrees in the cells. Compared with the model group, the red lipid droplet particles in the cells of the low, middle and high doses of traditional Chinese medicine and the positive medicine group decreased.

4.2 Results of intracellular TG/protein content

Compared with the blank group, the TG content of the cells in the model group was significantly increased, p<0.01, which was statistically significant.

Compared with the model group, the TG content in the cells of the high, middle and low dose groups of traditional Chinese medicine decreased, p<0.05, which was statistically significant, while the TG content in the cells of the positive drug group was significantly reduced, p<0.01, which was statistically significant.

Table 1 TG/protein content

Note: Compared with blank group *p<0.05, **p<0.01; compared with model group ^# p<0.05, ^## p<0.01

4.3 qPCR method to detect the expression results of target genes (TLR4, NF-κB, TGF-β1 and Smad3) at the mRNA level in HepG2 cells

Compared with the blank group, the expression levels of target genes TLR4, NF-κB, TGF-β1, and Smad3 in the model group were significantly up-regulated.

Compared with the model group, the expressions of TLR4, NF-κB, TGF-β1, and Smad3 at the mRNA level were significantly down-regulated in the high-dose, middle-dose, low-dose, and positive drug groups.

5 Conclusion

After treating NAFLD cells with drug-containing serum for 24 hours, the results of oil red O staining, TG/protein content, and qPCR assay showed that compared with the model group, the Chinese medicine group could reduce the TG content in NAFLD cells, possibly by down-regulating the target gene TLR4 , The expression of NF-κB at the mRNA level plays a role in the treatment of NAFLD. Moreover, the Chinese medicine group can also down-regulate the mRNA expression of TGF-β1 and Smad3 in the cells to prevent liver fibrosis.

The pharmaceutical composition of the invention has precise compatibility, and the flavors of the medicines complement each other, has clear curative effect on fatty liver, can be used to prevent liver fibrosis, has little toxic and side effects, and provides a new choice for clinical treatment of fatty liver.

Claims (10)

1. a kind of pharmaceutical composition, it is characterised in that：It is the preparation that the raw material matched by following weight is prepared from：

10-12.5 parts of barrenwort, 15-20 parts of the Radix Astragali, 5-7.5 parts of Poria cocos, 5-7.5 parts of the bighead atractylodes rhizome, 7.5-10 parts of rhizoma alismatis, dried orange peel 7.5- 10 parts, 7.5-10 parts of the tuber of pinellia, 15-20 parts of raw hawthorn, 15-20 parts of the red sage root.

2. pharmaceutical composition according to claim 1, it is characterised in that：It is the raw material matched by following weight prepare and Into preparation：

10 parts of barrenwort, 15 parts of the Radix Astragali, 7.5 parts of Poria cocos, 7.5 parts of the bighead atractylodes rhizome, 10 parts of rhizoma alismatis, 10 parts of dried orange peel, 7.5 parts of the tuber of pinellia, raw mountain 15 parts of short, bristly hair or beard, 15 parts of the red sage root.

3. pharmaceutical composition according to claim 1 or 2, it is characterised in that：It is the primary medicinal powder by the bulk drug, Or water or extractive with organic solvent are active component, add pharmaceutically acceptable auxiliary material or complementary composition is prepared from Preparation.

4. pharmaceutical composition according to claim 3, it is characterised in that：The preparation is oral formulations.

5. pharmaceutical composition according to claim 4, it is characterised in that：The oral formulations are granule, tablet, capsule Agent, oral liquid.

6. a kind of preparation method for preparing Claims 1 to 5 any one described pharmaceutical composition, it is characterised in that：It includes Following operating procedure：

(1) raw material of each weight proportion is weighed；

(2) beat powder or add water or organic solvent extraction, add pharmaceutically acceptable auxiliary material or complementary composition is prepared from Preparation.

7. purposes of the pharmaceutical composition in the medicine for preparing treatment fatty liver described in Claims 1 to 5 any one.

8. purposes according to claim 7, it is characterised in that：Described medicine is the medicine for treating NASH Thing.

9. purposes of the pharmaceutical composition in the medicine for preparing prevention liver fibrosis described in Claims 1 to 5 any one.

10. Claims 1 to 5 any one described pharmaceutical composition is in preparing with warming Yang, enlivening spleen, the medicine of clearing damp resolving sputum Purposes.