CN116813459B - Extraction method and application of wild buckwheat rhizome terpenoid - Google Patents

Abstract

The invention discloses an extraction method of wild buckwheat rhizome terpenoid, which comprises the following steps: 1) Reflux extracting rhizoma Fagopyri Dibotryis powder in ethanol solution for 3-5 times to obtain filtrate, and recovering ethanol to obtain extract; 2) Mixing the extract obtained in the step 1) with water, extracting with petroleum ether to obtain a petroleum ether layer, separating, and drying to obtain the wild buckwheat rhizome terpenoid. While providing its use. The invention separates the compound of the haematopoietic ketone and the haematopoietic alcohol by the repeated reflux extraction of the ethanol solution, and the method has high content of the extracted compound, simple method and easy popularization and application.

Description

Extraction method and application of golden buckwheat terpenoid compounds

Technical Field

The invention belongs to the technical field of plant extracts, and particularly relates to an extraction method and application of fagopyrum truncatum terpenoid compounds.

Background technique

Golden buckwheat tastes slightly spicy and astringent, and is cool in nature. It enters the lung meridian, clears away heat and detoxifies, discharges pus and removes blood stasis, and strengthens the stomach and helps digestion. The chemical components in golden buckwheat mainly include flavonoids, terpenoid compounds, organic acids, and steroid compounds. Studies have shown that golden buckwheat has the effects of anti-platelet aggregation, lipid regulation and hypoglycemic, anti-oxidation, enhancing the phagocytic function of macrophages, and anti-aging. The latest research has found that golden buckwheat can improve the high viscosity of patients' blood and inhibit platelet aggregation. In addition, golden buckwheat tea can regulate the glucose and lipid metabolism of type 2 diabetic model mice, specifically by reducing fasting blood sugar, total cholesterol, and triglycerides. Fagopyrum truncatum extract can enhance the phagocytic function of mouse peritoneal macrophages. The study found that after feeding mice with Fagopyrum truncatum extract, the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) in the blood, liver and heart of mice were increased to varying degrees, and the content of lipid peroxidation product malondialdehyde (MDA) decreased, indicating that it inhibits the generation of lipid peroxidation products, plays a role in scavenging oxygen free radicals and resisting lipid peroxidation, and achieves anti-inflammatory and lipid-lowering effects. Fully exploring the effective ingredients in Fagopyrum truncatum has important theoretical and practical significance.

Summary of the invention

The present invention aims to provide a method for extracting terpenoid compounds from Fagopyrum truncatum. Providing the application of terpenoid compounds from Fagopyrum truncatum is another object of the present invention.

Based on the above purpose, the present invention adopts the following technical solutions:

A method for extracting terpenoid compounds from golden buckwheat comprises the following steps:

1) placing golden buckwheat flour in an ethanol solution for reflux extraction 3-5 times to obtain a filtrate, recovering ethanol to obtain an extract;

2) extracting the extract in step 1) with petroleum ether to obtain a petroleum ether layer, separating and drying the layer to obtain golden buckwheat terpene compounds.

In step 1), the concentration of the ethanol solution is 30-70%, and the reflux extraction is performed 3 times. The mass of the ethanol solution added in each reflux extraction is 2-4 times the mass of the golden buckwheat powder, and the reflux extraction time is 1-5h.

In step 1), golden buckwheat is taken and crushed into coarse powder to obtain golden buckwheat powder, first adding 3 times the amount of 50% ethanol solution, soaking for 4 hours, reflux extraction for 3 hours, filtering, and the filtrate is reserved; adding 3 times the amount of 50% ethanol solution to the filter residue, heating and reflux for 2 hours, filtering, and the filtrate is reserved; adding 2 times the amount of 50% ethanol solution to the filter residue, heating and reflux for 1 hour, filtering, and the filtrate is reserved, combining the filtrates of the above reflux extraction, reducing pressure to recover ethanol, and obtaining an extract.

Application of golden buckwheat terpenoid compounds obtained by extraction method in drugs for treating primary hyperlipidemia.

The medicine is a solid preparation.

When the drug is made into a solid preparation, auxiliary materials are added, and the auxiliary materials are one or a combination of two or more of microcrystalline cellulose, cross-linked polyvinylpyrrolidone, starch and talcum powder.

The method for preparing the drug into a solid preparation comprises the following steps: 100g of golden buckwheat terpene compounds and auxiliary materials are crushed into fine powder respectively, sieved, 8g of cross-linked polyvinylpyrrolidone, 26g of microcrystalline cellulose and 27g of starch are weighed, dry-mixed, 95% of ethanol is added as a binder, 4# sieve is wet-granulated, dried at 60-70°C until the moisture content of the particles is 2.0-4.0%, 1% of talcum powder by weight of the particles is added, 4# sieve is used to size the particles, tabletted, and packaged in aluminum foil and PVC plastic blisters to obtain the solid preparation.

Preparation specifications: Each tablet weighs 0.4 mg. Usage: 4 tablets at a time, 3 times a day.

Compared with the prior art, the present invention has the following beneficial effects:

1) The present invention separates and obtains alderone and alder alcohol compounds by multiple reflux extraction with ethanol solution. The compound content extracted by the method is high, the method is simple, and it is easy to promote and apply;

2) Application of golden buckwheat terpenoids obtained by extraction method in the treatment of primary hyperlipidemia. After treatment with golden buckwheat terpenoid anti-inflammatory and lipid-lowering tablets, the SOD activity of mouse liver decreased, and the ROS level increased; the FTL-mRNA level increased significantly, indicating that golden buckwheat terpenoid anti-inflammatory and lipid-lowering tablets have obvious lipid-lowering effects and have a certain effect on lipid deposition in the liver of atherosclerosis model mice. The treatment of hypertension and the protective effect of golden buckwheat terpenoid anti-inflammatory and lipid-lowering tablets on aortic injury may be related to the inhibition of AKT/mTOR signaling pathway and autophagy. Golden buckwheat terpenoid anti-inflammatory and lipid-lowering tablets may inhibit autophagy through other signaling pathways.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solution of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without paying any creative work.

Figure 1 HE staining of mouse liver paraffin section, pathological morphology.

Detailed ways

In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention is described in detail below, but the following embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

Example 1

A method for extracting terpenoid compounds from golden buckwheat comprises the following steps:

1) Take golden buckwheat, crush it into coarse powder, and obtain golden buckwheat powder. Place the golden buckwheat powder in a 50% ethanol solution for reflux extraction 3 times, first add 3 times the amount of 50% ethanol solution, soak for 4 hours, reflux extraction for 3 hours, filter, and set the filtrate aside; add 3 times the amount of 50% ethanol solution, heat and reflux for 2 hours, filter, and set the filtrate aside; add 2 times the amount of 50% ethanol solution, heat and reflux for 1 hour, filter, and set the filtrate aside, combine the filtrates of the above reflux extraction, and reduce the pressure to recover ethanol to obtain an extract;

2) Extract step 1) with petroleum ether to obtain a petroleum ether layer, separate it by silica gel column chromatography, and use a petroleum ether-acetone system gradient elution on the silica gel column after loading, recover the organic solvent in the eluate, and dry it to obtain golden buckwheat terpene compounds. The silica gel column after loading is first eluted with a petroleum ether-acetone mixed solvent with a volume ratio of 100:1, and then eluted with a petroleum ether-acetone mixed solvent with a volume ratio of 10:3.

The terpenoids of golden buckwheat were dissolved in chloroform, mp: 245-2470C; the FeCl ₃ reaction was negative, indicating that the compound had no phenolic hydroxyl group, and the Li ebermann-Burchard reaction was positive. Further nuclear magnetic resonance (1H-NMR, 13C-NMR), combined with physicochemical properties and spectral data, it was found that the main active ingredients of the terpenoids of golden buckwheat were alderone and alder alcohol.

The invention discloses an application of golden buckwheat terpenoid compounds obtained by an extraction method in a drug for treating primary hyperlipidemia. The drug is a solid preparation and is added with excipients, which include microcrystalline cellulose, cross-linked polyvinylpyrrolidone, starch and talcum powder.

The method for preparing the drug into a solid preparation comprises the following steps: 100g of golden buckwheat terpene compounds and auxiliary materials are respectively crushed into fine powder, sieved, 8g of cross-linked polyvinylpyrrolidone, 26g of microcrystalline cellulose and 27g of starch are weighed, dry-mixed, 95% of ethanol is added as a binder, 4# sieve is wet-granulated, dried at 65°C until the moisture content of the particles is 2.0%, 1% of talcum powder by weight of the particles is added, 4# sieve is used to size the particles, tabletted, and packaged in aluminum foil and PVC plastic blisters to obtain the solid preparation.

Example 2

The difference from Example 1 is that the invention provides a method for extracting terpene compounds from golden buckwheat, comprising the following steps: 1) taking golden buckwheat, crushing it into coarse powder to obtain golden buckwheat powder, placing the golden buckwheat powder in a 60% ethanol solution for reflux extraction 3 times, first adding 3 times the amount of 60% ethanol solution, soaking for 4 hours, reflux extraction for 2 hours, filtering, and setting the filtrate aside; adding 2 times the amount of 60% ethanol solution, heating and refluxing for 2 hours, filtering, and setting the filtrate aside; adding 2 times the amount of 60% ethanol solution, heating and refluxing for 2 hours, filtering, and setting the filtrate aside; combining the filtrates of the above reflux extractions, recovering ethanol under reduced pressure, and obtaining an extract.

Example 3

The difference from Example 1 is that the invention provides a method for extracting terpene compounds from golden buckwheat, comprising the following steps: 1) taking golden buckwheat, crushing it into coarse powder to obtain golden buckwheat powder, placing the golden buckwheat powder in a 50% ethanol solution and reflux extracting it for 3 times, first adding 3 times the amount of 50% ethanol solution, soaking for 4 hours, reflux extracting for 4 hours, filtering, and setting the filtrate aside; adding 3 times the amount of 50% ethanol solution, heating and refluxing for 3 hours, filtering, and setting the filtrate aside; adding 3 times the amount of 50% ethanol solution, heating and refluxing for 2 hours, filtering, and setting the filtrate aside, combining the filtrates of the above reflux extractions, and recovering the ethanol under reduced pressure to obtain an extract.

Example 4 Test Example

The terpenoid components alderone and alder alcohol in Fagopyrum truncatum were used as content determination indicators, and the ethanol concentration, extraction time, extraction times and solvent dosage were used as investigation factors to screen the extraction process of Fagopyrum truncatum.

1. Effect of ethanol concentration

Take golden buckwheat, crush it into coarse powder, divide it into three groups, weigh three portions in each group, each portion is 1000g, add 6 times the amount of ethanol solution with concentration of 30%, 50%, and 70% respectively in each group, soak for 4 hours, then reflux and extract for 1 hour, filter, collect the filtrate, concentrate under reduced pressure, dry to 4% water content, get dry paste, weigh, and do 3 parallel tests in each group at the same time. Measure the content of alderone and alder alcohol in each group of dry paste, and the results are shown in Table 1 below.

Among them, the dry paste rate = the extracted dry paste powder (Fagopyrum truncatum terpenoid compounds)/the amount of crude drug used for extraction (Fagopyrum truncatum raw material) × 100%.

Table 1 Effect of ethanol concentration

It can be seen from Table 1 that the contents of alderone and alder alcohol in the 50% ethanol solution are relatively high.

2. Influence of extraction time

Take golden buckwheat, crush it into coarse powder, divide it into three groups, three portions in each group, weigh 1000g in each portion, add 6 times the amount of 50% ethanol solution to each group, soak for 4 hours, reflux extract for 3h, 1h, 2h respectively, filter, collect the filtrate, concentrate under reduced pressure, dry to 4% water content, get dry paste, weigh it, and do 3 parallel tests for each group at the same time. The results are shown in Table 2 below.

Table 2 Effect of extraction time

As shown in Table 2, when the extraction was performed for 2 h, the extraction amount of terpenoid compounds of Fagopyrum serrata was relatively high, which was beneficial to the extraction of effective ingredients in the medicinal materials.

3. Impact of extraction times

Take golden buckwheat, crush it into coarse powder, divide it into three groups, three parts in each group, weigh 1000g for each part, add 6 times the amount of 50% ethanol solution to test group 1, soak for 4h, and then reflux extract for 2h. Test group 2 refluxed twice, first added 3 times the amount of 50% ethanol solution, soaked for 4h, refluxed for 2h, and then added 3 times the amount of 50% ethanol solution, refluxed for 2h. Test group 3 refluxed for 3 times, added 3 times the amount of 50% ethanol solution, soaked for 4h, refluxed for 2h; then added 2 times the amount of 50% ethanol solution, refluxed for 2h; finally added 2 times the amount of 50% ethanol solution, refluxed for 2h, filtered, collected the filtrate, concentrated under reduced pressure, dried to 4% water content, obtained dry paste, weighed, and each group did 3 parallel tests at the same time. Measure the content of red ketone and red ketone alcohol in each group of dry paste and take the average value. The results are shown in Table 3 below.

Table 3 Effect of extraction times

As shown in Table 3, with the increase of extraction times, the dry extract rate and the amount of terpenoid compounds extracted from Fagopyrum truncatum increased. The increase of reflux extraction times was beneficial to the extraction of effective ingredients of Fagopyrum truncatum.

4. Influence of solvent dosage

Take golden buckwheat, grind it into coarse powder, divide it into three groups, each group has three portions, each portion weighs 1000g, and each group is tested three times in parallel at the same time:

In the first test group, after adding 3 times the amount of 50% ethanol solution, soak for 4 hours, reflux extraction for 2 hours, filter and collect the filtrate; after adding 2 times the amount of 50% ethanol solution, reflux extraction for 2 hours, filter and collect the filtrate; after adding 2 times the amount of 50% ethanol solution to the filter residue, reflux extraction for 2 hours, filter and collect the filtrate, combine the three filtrates, concentrate under reduced pressure, and dry to 4% moisture.

In the experiment group 2, after adding 3 times the amount of 50% ethanol solution, soak for 4 hours, reflux extraction for 2 hours, filter and collect the filtrate; after adding 3 times the amount of 50% ethanol solution, reflux extraction for 2 hours, filter and collect the filtrate; after adding 2 times the amount of 50% ethanol solution to the filter residue, reflux extraction for 2 hours, filter and collect the filtrate, combine the three filtrates, concentrate under reduced pressure, and dry to 4% moisture.

Three groups were tested. After adding 3 times the amount of 50% ethanol solution, soaking for 4 hours, reflux extraction for 2 hours, filtering with filter cloth, and collecting the filtrate; after adding 3 times the amount of 50% ethanol solution, reflux extraction for 2 hours, filtering with filter cloth, and collecting the filtrate; after adding 3 times the amount of 50% ethanol solution to the filter residue, reflux extraction for 2 hours, filtering with filter cloth, collecting the filtrate, combining the three filtrates, concentrating under reduced pressure, drying to 4% water content, obtaining a dry paste, and weighing. The results are shown in Table 4 below.

Table 4 Effect of solvent dosage

As shown in Table 4, through the investigation of the dosage of 50% ethanol solution, the first test group used 3 times, 2 times and 2 times the dosage for three extractions, the second test group used 3 times, 3 times and 2 times the dosage for three extractions, and the third test group used 3 times, 3 times and 3 times the dosage for three extractions. The total amount of ethanol solution used in the three groups of experiments was 7 times, 8 times and 9 times the dosage of the medicinal materials, respectively. With the increase of the amount of solvent, the terpene compounds of Fagopyrum truncatum did not show a significant increase trend.

Example 5 Clinical Trial

1. Materials

1.1 General Information

Patients with dyslipidemia from the Third Affiliated Hospital of Henan University of Traditional Chinese Medicine, Yanling County Hospital of Traditional Chinese Medicine and Changge Central Hospital from June 2018 to July 2020 were selected and divided into groups according to the principle of randomized, double-blind control, with 45 cases in each group. Control group: 25 males and 20 females, aged 34 to 68 years, BMI (24.44±3.12), course of disease 7 months to 5 years. Experimental group: 23 males and 22 females, aged 30 to 69 years, BMI (24.56±2.65), course of disease 8 months to 6 years. There was no difference in the basic clinical data of the two groups of patients, which was comparable (P＞0.05). See Table 5 for details.

Table 5 General clinical data

Note: Compared with the control group, P＞0.05

1.2 Reagents and instruments

TG kit (2021081201), TC kit (2021072702), LDL-C kit (2021110101) were all purchased from Anhui Ipronocon Company, HDL-C kit (20110815, Shanghai Fosun Changzheng Company), etc.

1.3 Diagnostic criteria

Formulated with reference to the "Guidelines for the Prevention and Treatment of Dyslipidemia in Adults in China (2016 Revised Edition)"; TCM Syndrome Differentiation Standards: Formulated according to the "Expert Consensus on the Diagnosis and Treatment of Dyslipidemia with Integrated Traditional Chinese and Western Medicine" (2017 Edition), the main symptoms are heavy head, dizziness, and numbness of the limbs. Secondary symptoms are fatigue, poor appetite, and tasteless mouth. Tongue and pulse: pale tongue; white, thick or greasy tongue coating; or stringy and slippery.

1.4 Inclusion criteria

① Meet the diagnostic criteria of Western medicine; ② Age between 30 and 70 years old (inclusive); ③ TCM diagnosis of phlegm and blood stasis syndrome; ④ Sign the informed consent form.

1.5 Exclusion criteria

① Those with secondary dyslipidemia; ② Those with other serious diseases such as serious cardiovascular and cerebrovascular diseases; ③ Those with abnormal liver and kidney function; ④ Those who are allergic to the drugs used in this study or have allergic constitution; ⑤ Pregnant and lactating women; ⑥ Those with poor compliance and those who drop out midway due to various factors.

2. Methods and Results

2.2 Clinical dosing regimen

The lead-in period after enrollment was 1 week, during which Chinese and Western medicines for the treatment of dyslipidemia were discontinued.

Test group: solid preparation of Example 1 of the present invention (Fagopyrum truncatum terpenoid anti-inflammatory and lipid-lowering tablets), 4 tablets/time, 3 times/day. Control group: Hedan tablets (Beijing Beida Weixin Biotechnology Co., Ltd., batch number: Z10950029, 0.3g/tablet), 2 tablets/time, 2 times/day. Continuous treatment for 6 weeks, efficacy evaluation was performed at 3w and 6w time points respectively. All enrolled patients received health education, low-salt and low-fat diet, and appropriate exercise.

2.3 Sample collection

Take 4 ml of forearm venous blood in the morning at 0w, 3w, and 6w in the fasting state (fasting for about 12h), put it into EDTA tube, centrifuge it, separate the upper layer of serum and store it in a -80℃ refrigerator for later use;

2.4 Observation indicators

2.4.1 Blood lipid measurement

Venous blood was collected at 0w, 3w, and 6w to measure serum TC, TG, LDL-C, and HDL-C levels.

2.4.2 TCM clinical symptoms and syndrome scores

The syndrome grading quantitative table was developed with reference to the Expert Consensus on the Diagnosis and Treatment of Dyslipidemia with Integrated Traditional Chinese and Western Medicine, including the four main symptoms of obesity, heaviness, chest tightness and palpitations, and limb numbness, which were scored as none, mild, moderate, and severe, with scores of 0, 2, 4, and 6 points respectively. Dizziness, abdominal distension, fatigue, and tastelessness were scored as 0, 1, 2, and 3 points respectively. The higher the score, the more severe the condition. The results are shown in Table 6 below.

Table 6 Comparison of TCM syndrome scores between the two groups of patients

Note: Compared with the group at 0w1 ⁾ P＜0.05, ²⁾ P＜0.01; compared with the control group after treatment3 ⁾ P＞0.05

As shown in Table 6, compared with the 0 week of this group, the TCM syndrome scores of the two groups of patients at 3 weeks and 6 weeks were significantly reduced (P<0.05). Compared with the control group after treatment, there was no statistically significant difference in the TCM syndrome scores between the two groups at the same time point (P>0.05).

2.4.3 Safety indicators

① Safety indicators such as vital signs, blood routine, liver and kidney function, urine and stool routine before and after treatment. ② Adverse reactions during treatment, judge and deal with them.

Three patients in the control group had mild abnormal liver function, and one patient in the experimental group had mild constipation, with mild symptoms. No adverse reactions occurred in the other patients.

2.5 Criteria for determining efficacy

Refer to the "Guidelines for Clinical Research of New Chinese Medicines". Markedly effective: Patients whose blood lipid test reaches any of the following: TC-LDL-C/LDL-C decreases ≥20%, TC decreases ≥20%, TG decreases ≥40%, and HDL-C increases ≥0.26mmol/L. Effective: Patients whose blood lipid test reaches any of the following: TC-LDL-C/LDL-C decreases ≥10%, but <20%, TC decreases ≥10%, but <20%, TG decreases ≥20%, but <40%, and HDL-C increases ≥0.104mmol/L, but <0.26mmol/L. Invalid: Patients whose blood lipid test does not meet the above standards. The results are shown in Tables 7 and 8.

Total effective rate = (number of markedly effective + effective) cases/total number of cases × 100%.

Table 7 Changes in blood lipid levels at different time points (n=30, )mmol/L

Note: Compared with the group at 0 week ¹⁾ P <0.05; compared with the control group after treatment ³⁾ P > 0.05

Table 8 Comparison of clinical efficacy between the two groups of patients

Note: Compared with the control group1 ⁾ P＞0.05

As shown in Table 7, there was no significant difference in TC, LDL-C, TG, and HDL-C levels between the two groups before treatment (P>0.05). Compared with the group before treatment, the levels of TC, TG, and LDL-C in the two groups at 3 weeks and 6 weeks were significantly reduced (P<0.05), and HDL-C increased at 6 weeks. Compared with the control group after treatment, there was no statistically significant difference between the two groups (P>0.05).

As shown in Table 8, after treatment, the total effective rate of the control group was 89.66%, and the total effective rate of the experimental group was 83.33%, and there was no statistically significant difference between the two groups (P>0.05).

2.6 Statistical methods

SPSS 20.0 software was used for statistical analysis. Quantitative data that conformed to or were approximately normally distributed were expressed as x±s and analyzed by repeated measures analysis of variance. Those that did not conform to a normal distribution were described by quartiles and nonparametric tests were used.

Research results: The solid preparation prepared by the present invention - Golden Fagopyrum Terpenoid Anti-inflammatory and Lipid-lowering Tablets can significantly reduce the TCM syndrome score, improve the clinical symptoms of patients, reduce the levels of TC, TG, and LDL-C in the patient's serum (P < 0.05), and increase the level of HDL-C (P < 0.05), and no obvious adverse reactions occur. It can be seen that Golden Fagopyrum Terpenoid Anti-inflammatory and Lipid-lowering Tablets are safe and effective in treating dyslipidemia, and can correct the abnormal blood lipids in patients.

Example 6 Effects of Fagopyrum truncatum Terpenoid Anti-inflammatory and Lipid-lowering Tablets on Lipid Deposition in the Liver of Atherosclerotic Mice 1. Materials

1.1 Animals

Twenty-four healthy male mice, 8 weeks old, weighing (20±2) g, were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., license number: SCXK(Beijing)2022-0001.

1.2 Drugs, reagents and instruments

Cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) assay kits were purchased from Shanghai Kehua Biotechnology Co., Ltd.; reduced glutathione (GSH), malondialdehyde (MDA), and superoxide dismutase (SOD) were purchased from Nanjing Jiancheng Bioengineering Institute Co., Ltd.; total antioxidant capacity (T-AOC) test kit was purchased from Biyuntian Biotechnology Co., Ltd.; FTH1antibody, GPX4 antibody, p53antibody, and xCT antibody were purchased from ABCLONE Company, and the fully automatic biochemical analyzer was (Hitachi 7180, Japan).

2. Methods and Results

2.1 Model preparation, grouping and intervention

24 mice were randomly divided into a model group, a group of the golden buckwheat terpenoid anti-inflammatory and lipid-lowering tablets of the present invention, and a positive control group, with 8 mice in each group, and 8 mice as a normal group. The normal group was given a basic feed every day. The atherosclerosis model was prepared by feeding mice with a high-fat diet, and a high-fat diet (containing 0.15% cholesterol and 21% fat) was fed every day for 8 weeks. After 8 weeks of modeling, the drug was administered by gavage, and the dosage was converted according to the conversion coefficient of the body surface area between humans and animals. The golden buckwheat terpenoid anti-inflammatory and lipid-lowering tablets group and the positive control group were given golden buckwheat terpenoid anti-inflammatory and lipid-lowering tablets (130 mg/kg/d) and simvastatin (2.275 mg/kg/d) by gavage for 4 weeks, respectively, and the normal group and the model group were given an equal volume of normal saline by gavage for 4 weeks.

2.1 Blood lipid testing

The mice were fasted but not watered for 12 hours before sampling. The mice were weighed on the day of sampling and anesthetized with 10% chloral hydrate. Blood was collected from the abdominal aorta, allowed to stand at room temperature, centrifuged, and stored at -20°C for later use. The TC, TG, LDL-C, and HDL-C levels in the mouse serum were detected by an automatic biochemical analyzer. The results are shown in Table 9.

Table 9 Comparison of serum TG, TC, LDL-C, and HDL-C levels in each group of mice ( n＝9)

Note: Compared with the normal group, ^* P<0.05; compared with the model group, ^▲ P<0.05.

As shown in Table 9, compared with the normal group, the TG, TC, and LDL-C levels of the mice in the model group were significantly increased, and the HDL-C level was significantly decreased (P<0.05); compared with the model group, the simvastatin group and the golden buckwheat terpenoid anti-inflammatory and lipid-lowering tablets group could significantly reduce the TG, TC, and LDL-C levels in the serum of the model control group, and the simvastatin group and the golden buckwheat terpenoid anti-inflammatory and lipid-lowering tablets group could significantly reduce the TG, TC, and LDL-C levels in the serum of the model group, and the simvastatin group was superior to the golden buckwheat terpenoid anti-inflammatory and lipid-lowering tablets group (P<0.05), and the HDL-C level in the serum of the simvastatin group and the golden buckwheat terpenoid anti-inflammatory and lipid-lowering tablets group was significantly increased (P<0.02).

2.3 HE staining

After blood was collected, the aorta was removed, fixed with paraformaldehyde, embedded in paraffin, and sliced according to the instructions of the kit. Microscope examination and image acquisition and analysis were performed.

2.4 TUNEL staining

According to the instructions of the kit, the slides were sealed with anti-fluorescence quenching sealing agent, and the images were observed and collected under a fluorescence microscope.

2.4 HE staining to observe liver pathological morphology

Mouse liver tissue was fixed in 4% paraformaldehyde for 72 hours, and stained with hematoxylin-eosin (HE) according to the conventional method, dehydrated with 70-100% gradient ethanol, transparentized with xylene, embedded in paraffin, sliced, embedded in paraffin, and the mouse liver tissue morphology was observed under an optical microscope, as shown in Figure 1.

Among them, in Figure 1, A. normal group (200×), B. model group (200×), C. simvastatin group (200×), D. golden buckwheat terpenoid anti-inflammatory and lipid-lowering tablets group (200×).

As shown in Figure 1, the normal group had normal liver lobule structure, plump cell morphology, and no obvious lipid droplets in the cytoplasm; the model group had enlarged liver cells, a large number of fat vacuoles in the cytoplasm, and the liver sinusoids were narrowed or disappeared. The simvastatin group had significantly fewer lipid droplets, and its pathological changes were significantly alleviated compared with the model group; the recovery degree of the golden buckwheat terpenoid anti-inflammatory and lipid-lowering tablet group was similar to that of the simvastatin group, the degree of fatty degeneration of liver cells was alleviated, and the number of fat vacuoles was significantly reduced.

2.5 Detection of SOD activity and GSH levels in each group of mice

According to the instructions of the kit, the total antioxidant capacity of serum was determined by ABTS rapid method, the SOD activity in serum was detected by WST-1 method, and the GSH expression in serum was detected by spectrophotometry. See Table 10.

Table 10 Changes in serum ROS and SOD activity in each group of mice ( n＝9)

Note: Compared with the normal group, ^** P<0.05; compared with the model group, ^▲▲ P<0.05.

As shown in Table 10, compared with the normal group, the ROS content in the model group was increased (P<0.05), and the SOD activity was decreased (P<0.05); compared with the model group, the ROS in the simvastatin group and the golden buckwheat terpenoid anti-inflammatory and lipid-lowering tablets group was significantly decreased (P<0.05), the GSH content was increased (P<0.05), and the SOD activity was significantly increased (P<0.05).

2.5 RT-PCR detection of liver tissue-related mRNA expression levels

According to the kit instructions, the total RNA concentration of the sample was diluted to 200 ng/μL and then reverse transcribed into cDNA, added to the amplification reaction system, and the real-time fluorescence quantitative PCR method was used. The △△CT method was used for relative quantitative analysis. The primers were synthesized by Wuhan Jinkairui Bioengineering Co., Ltd., and the sequences are shown in Table 11. The expression level of mouse liver mRNA is shown in Table 12.

Table 11 Primer sequence list

Table 12 mRNA expression levels in mouse liver

Note: Compared with the normal group, ^* P<0.05; compared with the model group, ^▲ P<0.05.

As shown in Table 12, compared with the normal group, the mRNA expressions of PTGS2 and NOX1 in the liver tissue of mice in the model group were significantly decreased (P<0.05), and the mRNA expression of p53 was significantly increased (P<0.05); compared with the model group, the mRNA expressions of PTGS2 and NOX1 in the liver of mice in the simvastatin group and the golden buckwheat terpenoid anti-inflammatory and lipid-lowering tablets group were increased (P<0.05), and the mRNA expression of p53 was significantly decreased (P<0.05).

Compared with the model group, after treatment with golden buckwheat terpenoid anti-inflammatory and lipid-lowering tablets and simvastatin, the SOD activity in the liver of mice decreased, and the ROS level increased; the FTL-mRNA level increased significantly, indicating that golden buckwheat terpenoid anti-inflammatory and lipid-lowering tablets have a significant lipid-lowering effect and have a certain effect on lipid deposition in the liver of atherosclerosis model mice. The treatment of hypertension and the protective effect of golden buckwheat terpenoid anti-inflammatory and lipid-lowering tablets on aortic injury may be related to the inhibition of AKT/mTOR signaling pathway and autophagy. Golden buckwheat terpenoid anti-inflammatory and lipid-lowering tablets may inhibit autophagy through other signaling pathways.

2.6 Statistical analysis

The experimental data were all quantitative data, and SPSS19.0 software was used for statistical analysis. (x±s) was used to represent the difference between multiple groups. One-way analysis of variance was used for comparison, and P<0.05 represented statistical significance.

Claims (5)

1. The use of golden buckwheat terpenoids in the preparation of drugs for treating primary hyperlipidemia, characterized in that the extraction of the golden buckwheat terpenoids comprises the following steps: 1) Place golden buckwheat flour in an ethanol solution for reflux extraction 3-5 times to obtain a filtrate, recover ethanol, and obtain an extract; 2) extracting the extract of step 1) with petroleum ether to obtain a petroleum ether layer, separating and drying it to obtain golden buckwheat terpenoid compounds alderone and alder alcohol; In step 1), the concentration of the ethanol solution is 30-70%, and the reflux extraction is performed 3 times. The mass of the ethanol solution added in each reflux extraction is 2-4 times the mass of the golden buckwheat powder, and the reflux extraction time is 1-5h; In step 2), the extract of step 1) is extracted with petroleum ether to obtain a petroleum ether layer, which is separated by silica gel column chromatography. The silica gel column loaded with the sample is gradient eluted with a petroleum ether-acetone system, and the organic solvent in the eluate is recovered and dried to obtain golden buckwheat terpene compounds alderone and alder alcohol; In step 2), the silica gel column after loading is first eluted with a petroleum ether-acetone mixed solvent with a volume ratio of 100:1, and then eluted with a petroleum ether-acetone mixed solvent with a volume ratio of 10:3. 2. The use according to claim 1, characterized in that the medicine is a solid preparation. 3. The use according to claim 2, characterized in that an excipient is added when the drug is made into a solid preparation, and the excipient is one or a combination of two or more of microcrystalline cellulose, cross-linked polyvinylpyrrolidone, starch, and talc. 4. The use according to claim 3, characterized in that the method for preparing the drug into a solid preparation is: 100g of golden buckwheat terpene compounds and auxiliary materials are crushed into fine powder respectively, sieved, 8g of cross-linked polyvinylpyrrolidone, 26g of microcrystalline cellulose, and 27g of starch are weighed, dry-mixed, 95% ethanol is added as a binder, and wet granulation is performed with a 4# sieve. The particles are dried at 60-70°C until the moisture content of the particles is 2.0-4.0%, 1% of talcum powder by mass of the particles is added, granulated with a 4# sieve, tableted, and packaged in aluminum foil and PVC plastic blisters to obtain the product. 5. The use as claimed in claim 1, characterized in that, in step 1), golden buckwheat is taken, crushed into coarse powder to obtain golden buckwheat powder, first added with 3 times the amount of 50% ethanol solution, soaked for 4 hours, refluxed for extraction for 3 hours, filtered, and the filtrate is reserved; the filter residue is added with 3 times the amount of 50% ethanol solution, heated and refluxed for 2 hours, filtered, and the filtrate is reserved; the filter residue is added with 2 times the amount of 50% ethanol solution, heated and refluxed for 1 hour, filtered, and the filtrate is reserved, the filtrates of the above reflux extractions are combined, and the ethanol is recovered under reduced pressure to obtain an extract.