阅读说明：本技术 一种水红花子提取物在制备预防或治疗非酒精性脂肪肝疾病药物中的应用 (Application of fructus Polygoni orientalis extract in preparing medicine for preventing or treating non-alcoholic fatty liver disease ) 是由 栗政 陈凯 李进冬 周磊 陈艳 花慧莲 李建华 瞿建江 于 2021-07-22 设计创作，主要内容包括：本发明提供了水红花子提取物作为唯一活性成分在制备预防或治疗非酒精性脂肪肝疾病药物中的应用。本发明中,水红花子提取物主要含有黄酮和多酚酸类化合物,能有效缓解游离脂肪酸诱导的人肝细胞中脂质蓄积,降低甘油三酯和胆固醇水平,降低氧化应激和炎症因子水平,说明水红花子提取物能对非酒精性脂肪肝起到显著的治疗作用,可用于制备预防或治疗非酒精性脂肪肝疾病药物。(The invention provides application of a fructus polygoni orientalis extract as a unique active ingredient in preparing a medicament for preventing or treating non-alcoholic fatty liver diseases. In the invention, the fructus polygoni orientalis extract mainly contains flavone and polyphenolic acid compounds, can effectively relieve lipid accumulation in human liver cells induced by free fatty acid, reduce triglyceride and cholesterol levels, and reduce oxidative stress and inflammatory factor levels, which indicates that the fructus polygoni orientalis extract can play a significant role in treating non-alcoholic fatty liver and can be used for preparing medicaments for preventing or treating non-alcoholic fatty liver diseases.)

1. An application of fructus Polygoni orientalis extract as the only active ingredient in preparing medicine for preventing or treating non-alcoholic fatty liver disease is provided.

2. The use as claimed in claim 1, wherein said princesplume ladysthumb extract contains mainly the following compounds:

3. the use as claimed in claim 1, wherein the fructus Polygoni orientalis extract is prepared by extracting fructus Polygoni orientalis with organic solvent or water as extraction solvent by ultrasonic extraction or reflux extraction.

4. The use as claimed in claim 3, wherein the extract of princesplume ladysthumb is prepared by extracting with ethanol under ultrasonic conditions.

5. The use as claimed in claim 4, wherein the princesplume ladysthumb extract is prepared by the steps of: the method comprises the following steps of crushing dry polygonum orientale medicinal materials, soaking in 80% ethanol, carrying out ultrasonic extraction, combining extracting solutions, filtering, recovering a solvent, and concentrating to obtain a polygonum orientale extract, wherein the mass volume ratio of the dry polygonum orientale medicinal materials to the 80% ethanol is 1 g: 2 mL.

6. The pharmaceutical composition is characterized in that the pharmaceutical composition takes a fructus polygoni orientalis extract as the only active ingredient, and the pharmaceutical composition comprises the fructus polygoni orientalis extract with effective amount in prevention or treatment and pharmaceutically acceptable auxiliary materials or carriers.

7. The pharmaceutical composition as claimed in claim 6, wherein the princesplume ladysthumb extract contains mainly the following compounds:

8. the pharmaceutical composition of claim 6, wherein the pharmaceutically acceptable excipient or carrier comprises one or more of a binder, a lubricant, a disintegrant, a cosolvent, a diluent, a stabilizer, a suspending agent, and a matrix.

9. The pharmaceutical composition of claim 6, wherein the pharmaceutically acceptable excipient or carrier is selected from one or more of starch, dextrin, sucrose, lactose, vegetable oil, microcrystalline cellulose, hydroxypropyl methylcellulose, low-substituted hydroxypropyl cellulose, crospovidone, sodium hydroxymethyl starch, polyethylene glycol, polyvinylpyrrolidone, magnesium stearate, silca gel, glucose, mannitol, and xylitol.

10. The pharmaceutical composition of claim 6, wherein the pharmaceutical composition is in the form of granules, tablets, hard capsules, soft capsules, drops, oral liquid, aerosols, nasal drops or injections.

Technical Field

The invention relates to a new application of a Fructus Polygoni Orientalis (Polygoni oriental lis Fructus) extract, in particular to a new application of the Fructus Polygoni Orientalis extract in the pharmaceutical field, and specifically relates to an application of the Fructus Polygoni Orientalis extract in preparing a medicament for preventing or treating non-alcoholic fatty liver diseases.

Background

Non-alcoholic fatty liver disease (NAFLD) is a metabolic stress liver injury closely associated with obesity, and its spectrum of disease includes progression from Non-alcoholic liver steatosis to Non-alcoholic steatohepatitis (NASH), liver fibrosis and cirrhosis, ultimately leading to hepatocellular carcinoma. With the increase in obesity, insulin resistance and metabolic syndrome, NAFLD has become the most common liver disease in developed countries, affecting approximately one third of the world's population, being one of the huge and growing disease burdens currently facing the world. In China, NAFLD has become the leading cause of abnormality of biochemical indexes of the first chronic liver disease and health physical examination liver. Besides affecting liver, NAFLD is also closely related to the onset of type 2 diabetes, cardiovascular and cerebrovascular diseases, and chronic kidney disease. For this reason, NAFLD has become a new challenge in the contemporary medical field, seriously jeopardizing human life health, and therefore the exploration of NAFLD preventive and therapeutic strategies has been reluctant.

Currently, the prevention and treatment measures for NAFLD mainly include lifestyle changes and drug therapy. Lifestyle changes centered on dietary intervention and exercise are effective in controlling/reversing simple NAFLD and are therefore generally recommended for use in NAFLD primary stage therapy. However, the change in lifestyle is difficult to maintain for a long time and difficult to implement, and thus the desired effect is often not achieved. Moreover, it has no obvious curative effect on hepatic fibrosis patients. Currently, there is no approved therapeutic drug for NAFLD. Clinically, hypoglycemic agents such as metformin, pioglitazone, hypolipidemic agents such as fenofibrate, antioxidant supplements such as vitamin E are recommended for the treatment of NAFLD. However, these drugs have limited use because of side effects, increased liver burden, or poor drug efficacy. Therefore, the development of safe and effective therapeutic drugs and health care products has important innovative significance and clinical value.

Fructus Polygoni Orientalis (POF) is dried mature fruit of Polygonum tinctorium L. Red knotweed is an annual herb plant in Polygonum of Polygonaceae, and is widely distributed in most of Jiangsu, Shandong, Hebei, Shanxi and other provinces in China. The red knotweed leaves are green and dense in flowers, and the fruits are used as medicines, so the red knotweed is a common plant for both ornamental and medicinal purposes. The fructus polygoni orientalis is salty and slightly pungent in flavor and slightly cold in nature, enters liver and stomach meridians, and has the functions of dissipating blood stasis, eliminating mass, removing food retention, relieving pain, inducing diuresis and relieving swelling and the like. In recent years, researchers at home and abroad have studied the components and biological activity of the princesplume ladysthumb. Research results show that the fructus polygoni orientalis has various beneficial pharmacological effects and health-care effects, including the effects of relieving acute liver injury, resisting inflammation, resisting oxidation, regulating immunity, resisting tumors and the like. At present, the research on the fructus polygoni orientalis mostly focuses on the analysis and detection of flavone of the fructus polygoni orientalis, and the research on the treatment effect of the fructus polygoni orientalis extract on rat liver injury is also related, the existing patent relates to the preparation of a flavone compound in the fructus polygoni orientalis, but the research on the prevention and treatment of the fructus polygoni orientalis applied to NAFLD alone is not reported.

Chinese patent application CN201610316887.4 relates to a compound traditional Chinese medicine composition for treating non-alcoholic fatty liver disease and a preparation method and application thereof. The traditional Chinese medicine is characterized by comprising the following traditional Chinese medicines in parts by weight: 3-5 parts of radix curcumae, 4-6 parts of rhizoma atractylodis, 3-5 parts of semen cassiae, 3-5 parts of hawthorn, 2-4 parts of winged euonymus twig and 4-6 parts of fructus polygoni orientalis. The compound traditional Chinese medicine composition disclosed in the prior patent comprises radix curcumae, rhizoma atractylodis, semen cassiae, hawthorn and winged euonymus twig besides fructus polygoni orientalis, and does not disclose that the fructus polygoni orientalis in the compound traditional Chinese medicine composition can be independently used as an active ingredient for treating the non-alcoholic fatty liver disease.

The prior art also does not report that the fructus polygoni orientalis extract can be used as an active ingredient for treating the non-alcoholic fatty liver disease.

Disclosure of Invention

The technical problem solved by the invention is as follows: aiming at the current treatment status of NAFLD, a new application of the fructus polygoni orientalis extract is provided, namely the application of the fructus polygoni orientalis extract as the only active ingredient in preparing the medicine for preventing or treating the nonalcoholic fatty liver disease.

Through a large number of experimental researches, the invention unexpectedly discovers that the fructus polygoni orientalis extract serving as the only active ingredient can effectively treat human liver cells (LO) induced by free fatty acid by acting alone₂) The internal lipid accumulation, the triglyceride and cholesterol level in the liver cells are reduced, the oxidative stress in the liver cells and the inflammatory factor level in the liver cells are reduced, and a new safe and effective selection is provided for the prevention and treatment of NAFLD.

The technical scheme provided by the invention is as follows:

the first purpose of the invention is to provide the application of the fructus polygoni orientalis extract as the only active ingredient in preparing the medicine for preventing or treating the nonalcoholic fatty liver disease.

In the invention, the fructus polygoni orientalis extract mainly contains the following compounds:

in the invention, the preparation method of the fructus polygoni orientalis extract can use water as an extraction solvent, and adopts ultrasonic extraction or reflux extraction to obtain the extract; or extracting with organic solvent by ultrasonic extraction or reflux extraction to obtain extract; the extract can also be obtained by any existing traditional Chinese medicine extraction method.

In the invention, the preparation method of the fructus polygoni orientalis extract preferably takes ethanol as an extraction solvent and adopts ultrasonic extraction to obtain the extract.

In the present invention, the preparation method of the princesplume ladysthumb extract preferably comprises the following steps: the method comprises the following steps of crushing dry polygonum orientale medicinal materials, soaking in 80% ethanol, carrying out ultrasonic extraction, combining extracting solutions, filtering, recovering a solvent, and concentrating to obtain a polygonum orientale extract, wherein the mass volume ratio of the dry polygonum orientale medicinal materials to the 80% ethanol is 1 g: 2 mL.

The second purpose of the invention is to provide a pharmaceutical composition of a medicament for preventing or treating non-alcoholic fatty liver disease, the pharmaceutical composition takes the fructus polygoni orientalis extract as the only active ingredient, and the pharmaceutical composition comprises the fructus polygoni orientalis extract with effective amount in prevention or treatment and pharmaceutically acceptable auxiliary materials or carriers.

In the present invention, the pharmaceutically acceptable adjuvant or carrier is a common type available in the pharmaceutical field, and may include one or more of a binder, a lubricant, a disintegrant, a cosolvent, a diluent, a stabilizer, a suspending agent, a matrix, or the like.

In the invention, the pharmaceutically acceptable adjuvant or carrier is preferably selected from one or more of starch, dextrin, sucrose, lactose, vegetable oil, microcrystalline cellulose, hydroxypropyl methylcellulose, low-substituted hydroxypropyl cellulose, crospovidone, sodium hydroxymethyl starch, polyethylene glycol, polyvinylpyrrolidone, magnesium stearate, vitamin powder silica gel, glucose, mannitol and xylitol.

In the invention, the composition can be prepared into various pharmaceutically acceptable dosage forms according to the conventional process, such as granules, tablets, hard capsules, soft capsules, drops, oral liquid, aerosol, nasal drops or injection and the like.

In the present invention, the non-alcoholic fatty liver disease is any one of non-alcoholic simple fatty liver, non-alcoholic steatohepatitis and non-alcoholic cirrhosis.

The technical scheme of the invention has the following technical effects:

(1) the fructus polygoni orientalis extract provided by the invention mainly contains flavone, phenolic acid and phytosterol compounds. The invention is obtained by screening and verifying a large number of experiments: the fructus polygoni orientalis extract can be used for treating the nonalcoholic fatty liver disease NAFLD by single action.

(2) According to Chinese pharmacopoeia, fructus Polygoni orientalis has effects of dispelling blood stasis, eliminating abdominal mass, resolving food stagnation, relieving pain, inducing diuresis and relieving swelling. The reported water extract and alcohol extract of fructus Polygoni orientalis can be used for treating animal hepatic fibrosis, liver cirrhosis, hyperuricemia, immune liver injury, and hepatocarcinoma. However, no report is available on the use of fructus polygoni orientalis extract as the only active ingredient for treating NAFLD. Non-alcoholic fatty liver disease NAFLD is a clinical pathological syndrome which has no history of excessive drinking, is caused by fat accumulation in liver cells due to various reasons and is mainly characterized by liver cell steatosis and lipid accumulation, and is also a common clinical disease, and non-alcoholic fatty liver disease (NAFLD) is closely related to oxidative stress according to literature reports.

(3) In the pharmacological experimental research on the fructus polygoni orientalis extract, the invention surprisingly finds that the fructus polygoni orientalis extract can effectively improve the lipid accumulation of the liver cells by the single action; can effectively improve the lipid level of the liver cells and relieve liver cell damage caused by lipid accumulation; can effectively relieve oxidative stress in liver cells; the fructus polygoni orientalis extract can effectively relieve the inflammation of liver cells, and proves that the fructus polygoni orientalis extract can be used as the only active ingredient for preparing the medicine for preventing or treating the non-alcoholic fatty liver disease.

Drawings

FIG. 1 shows a liquid chromatography-mass spectrometry chromatogram of fructus Polygoni orientalis extract.

FIG. 2 shows the fatty acid-induced LO of fructus Polygoni orientalis extract₂Effects of intracellular lipid accumulation.

FIG. 3 shows the fatty acid-induced LO of fructus Polygoni orientalis extract₂The effects of cellular oxidative stress.

Detailed Description

EXAMPLE 1 preparation of princesplume ladysthumb extract

1. Preparation method of fructus Polygoni orientalis extract

Dried fructus Polygoni orientalis (purchased from Tongrentang, Kyoho, Beijing) is pulverized into powder by a pulverizer, 50g of the powder is weighed and soaked in 80% ethanol (100mL) for 2h, ultrasonic extraction is carried out at room temperature for 30min, and then the filtrate is collected by filtration. Ultrasonically extracting the filter residue again for 30min at room temperature, filtering and collecting the filtrate. The filtrates were combined and concentrated under reduced pressure to an oil.

2. Liquid chromatography-mass spectrometry analysis of fructus Polygoni orientalis extract

The obtained oil was dissolved in 80% methanol (5mL), then centrifuged at 15000 Xg for 15min, the supernatant was taken and filtered through a 0.22 μm nylon membrane filter, and the sample solution was analyzed by LC-MS. The chromatographic conditions for this experiment were: adopting a Sepax GP-C18 chromatographic column (2.1mm multiplied by 150mm,1.8 mu m), taking water containing 0.1 percent formic acid as a mobile phase A and acetonitrile as a mobile phase B; the flow rate was 0.2mL/min, the column temperature was 35 ℃ and the following gradient elution procedure was used: 0-7-15-20-25-30-40min, 20% -30% -40% -50% -55% -65% -20% B, and the sample amount is 5 mu L. The mass spectrum conditions are as follows: the Q-TOF mass spectrum is provided with an electrospray ion source, the ion source adopts a negative ion mode, the ion spray voltage is 3000V, and the evaporator temperature is 280 ℃; sheath gas pressure 50 psi; the capillary temperature is 320 ℃, the auxiliary gas pressure is 15psi, the full sweep mode covers the range of m/z 100-. By matching the resulting chromatographic peak retention time, exact molecular ion mass and product ion to the corresponding chemical standards and literature data, 30 major chemical components were identified (fig. 1, table 1).

TABLE 1 major chemical components of fructus Polygoni orientalis extract

Example 2 improving effect of princesplume ladysthumb extract on NAFLD

1. Materials and reagents

Sodium oleate, sodium palmitate, oil red O, Bovine Serum Albumin (BSA) and Thiazolidinium bromide (MTT) were purchased from Sigma Aldrich, human hepatocyte line LO₂The cells were purchased from ATCC, the 1640 medium for cell culture, the penicillin-streptomycin double antibody solution and Fetal Bovine Serum (FBS) were purchased from GIBCO Invitrogen, the Triglyceride (TG), Total Cholesterol (TC), glutamic-pyruvic transaminase (ALT) and glutamic-oxaloacetic transaminase (AST) detection kit was purchased from Nanjing Biotechnology institute, the active oxygen fluorescent probe CM-H2DCFA was purchased from Molecular Probes, and the inflammatory factors TNF-alpha, IL-1 beta and IL-6 gene primers were purchased from Waals Dagen.

2. Experimental methods

LO₂Cells were cultured in 1640 medium (containing 10% FBS, 100U/mL penicillin and 100. mu.g/mL streptomycin) and placed in a 37 ℃ incubator supplied with 5% CO₂. The cells were divided into control group (NC), model group (FFA) and control group (POF high, middle and low three dose groups) of the princesplume ladysthumb extract prepared in example 1 after cell attachment, the culture solution of the control group was changed for 1640 cells, the model group and the administration group cells were changed for 1640 containing 1mM fatty acid (molar ratio of sodium oleate to sodium palmitate is 2:1) to induce NAFLD model of the cells, the cells of the administration group were added with the extract of example 1 (final concentration is 5,10and 20. mu.g/mL, respectively), and the cells were cultured for 24 hours.

After the cell treatment is finished, adopting oil red O solution to stain and evaluate the accumulation degree of intracellular lipid droplets, detecting the contents of TG, TC, ALT and AST in the cells according to the instruction of a kit, and adopting CM-H₂DCFA fluorescent probe detects intracellular reactive oxygen species level, and RT-QPCR is adopted to detect intracellular mRNA expression levels of TNF-alpha, IL-1 beta and IL-6.

3. Results

3.1 lipid accumulation in hepatocytes

As shown in FIG. 2, the accumulation of lipid droplets in cells was significantly increased in FFA group compared to NC group, while in POF group, the accumulation of lipid droplets in cells was significantly decreased in high dose POF group compared to FFA group, indicating that the extract of princesplume ladysthumb was effective in improving the accumulation of lipid in liver cells.

3.2 Biochemical indicators in hepatocytes

As shown in table 2, compared with the NC group, the FFA group had significantly increased levels of TG, TC, and ALT in cells, whereas the POF group had significantly decreased levels of TG, TC, and ALT in cells of the high-dose POF group, significantly decreased levels of TC and ALT in cells of the medium-dose POF group, significantly decreased levels of ALT in cells of the low-dose POF group, and dose-dependent pharmacological effects. The above results show that the fructus Polygoni orientalis extract can effectively improve the lipid level of the liver cells and relieve the liver cell damage caused by lipid accumulation.

TABLE 2 Biochemical index measurement results in hepatocytes

^***p<0.001 compared to control group;^#p<0.05,^##p<0.01,^###p<0.001 vs. model group

3.3 evaluation of oxidative stress in hepatocytes

As shown in FIG. 3, the intracellular reactive oxygen species level in the FFA group was significantly increased compared to the NC group, while the intracellular reactive oxygen species level in the POF group was significantly decreased in the POF group compared to the FFA group, indicating that the princesplume ladysthumb extract was effective in relieving oxidative stress in the hepatocytes.

3.4 levels of inflammatory factors in hepatocytes

As shown in Table 3, the expression levels of mRNA for TNF-alpha, IL-1 beta and IL-6 were significantly increased in the FFA group cells compared to the NC group; compared with the FFA group, the mRNA expression levels of TNF-alpha, IL-1 beta and IL-6 in the cells of the high-dose and medium-dose POF groups are obviously reduced, the mRNA expression levels of TNF-alpha and IL-6 in the cells of the low-dose POF groups are obviously reduced, and the pharmacological effect is dose-dependent. The results show that the fructus polygoni orientalis extract can effectively relieve the inflammation of the liver cells.

TABLE 3 intracellular mRNA expression levels of TNF- α, IL-1 β and IL-6

^***p<0.001 compared to control group;^#p<0.05,^##p<0.01,^###p<0.001 vs. model group

The invention is not limited to the specific technical solutions described in the above embodiments, and all technical solutions formed by equivalent substitutions are within the scope of the invention as claimed.