CN118304345A

CN118304345A - Lindera root extract for treating hyperlipidemia and preparation method and application thereof

Info

Publication number: CN118304345A
Application number: CN202410319435.6A
Authority: CN
Inventors: 叶合; 楼招欢; 赖威旨; 王盈盈; 黄建波; 杨小虎
Original assignee: Zhejiang University Of Traditional Chinese Medicine Songyang Research Institute Co ltd; Zhejiang Hospital
Current assignee: Zhejiang University Of Traditional Chinese Medicine Songyang Research Institute Co ltd; Zhejiang Hospital
Priority date: 2024-03-19
Filing date: 2024-03-19
Publication date: 2024-07-09

Abstract

The invention provides a lindera root extract for treating hyperlipidemia and a preparation method and application thereof, belonging to the technical field of biological medicine. The preparation method of the lindera root extract comprises the following steps: soaking radix Linderae in water, boiling, and condensing and refluxing to obtain medicinal liquid, i.e. radix Linderae extract. The lindera root extract can reduce serum TC, TG, LDL-C level of mice with hyperlipidemia and improve lipid deposition of liver tissues; the lindera root extract can effectively reduce serum cholesterol level, and up-regulate liver tissue ABCA1, ABCG1, PPARα, LXRα and FXR expression level related to the reverse cholesterol transport process; the Linderae radix extract has effects of inhibiting the uptake of cholesterol and ox-LDL by macrophages and reducing the lipid deposition of macrophages. The lindera root extract can endow the traditional qi-regulating medicine with new scientific connotation, and promote the development of new products of special blood lipid-lowering traditional Chinese medicines and the development and utilization of the lindera root.

Description

Lindera root extract for treating hyperlipidemia and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a lindera root extract for treating hyperlipidemia, and a preparation method and application thereof.

Background

Hyperlipidemia (HYPERLIPIDEMIA, HLP) generally refers to elevated levels of total cholesterol and/or triglycerides in serum, including various dyslipidemias of low high density lipoprotein cholesterol. Dyslipidemia characterized by high cholesterol levels is an important risk factor for atherosclerotic cardiovascular disease (ASCVD) such as coronary heart disease. How to effectively prevent and control the hyperlipidemia so as to reduce the incidence rate of ASCVD is a great social problem currently faced.

At present, the blood lipid reducing drugs commonly used clinically mainly comprise statin, fibrate and other chemical drugs, and the blood lipid reducing effect of the chemical drugs is definite, but adverse reactions with different degrees such as liver and kidney injury, gastrointestinal tract reaction and the like exist in long-term application. The traditional Chinese medicine has the characteristics of multiple targets, overall synergy and the like, and has unique advantages in the treatment of chronic metabolic diseases such as hyperlipidemia and the like. Research on the mechanism of action of the traditional Chinese medicine for reducing blood fat with definite curative effect is further developed, and research and development of novel traditional Chinese medicine for resisting hyperlipidemia are facilitated.

The radix Linderae is dried root tuber of radix Linderae LINDERA AGGREGATE (Sims) Kosterm of Lauraceae, and is one of new Zhejiang eight-ingredient cultivation varieties. The combined spicebush root is rich in components such as combined spicebush root etherlactone, isoquinoline alkaloid and the like, is a clinically common qi-regulating traditional Chinese medicine, and has good gastrointestinal function regulating effect. Whereas the use of the black-bone aqueous extract for the treatment of hyperlipidemia has been less studied.

Disclosure of Invention

Therefore, the invention aims to provide the lindera root extract for treating the hyperlipidemia, and the preparation method and the application thereof, wherein the lindera root extract can effectively reduce the blood lipid level of animals with hyperlipidemia models, and has good blood lipid reducing effect.

In order to achieve the above object, the present invention provides the following technical solutions:

The invention provides a preparation method of a lindera root extract, which comprises the following steps: soaking radix Linderae in water, boiling, condensing, refluxing, and filtering to obtain medicinal liquid, namely radix Linderae extract; the time of condensation reflux is 0.5-1.5 h.

Preferably, the mass ratio of the combined spicebush root to the water is 1:9-11, and the soaking time is 25-30 min.

Preferably, the method further comprises the following steps of: mixing the residues with water, heating and boiling, condensing and refluxing for 40-50 min, and combining the liquid medicine; the mass ratio of the dregs to the water is 1:7-9.

The invention also provides the lindera root extract obtained by the preparation method.

The invention also provides application of the lindera root extract in preparing a medicine for treating hyperlipidemia.

Preferably, the lindera root extract can reduce serum total cholesterol, triglyceride and low density lipoprotein cholesterol level.

Preferably, the lindera extract can up-regulate the expression level of ABCA1, ABCG1, pparα, lxrα and FXR in liver tissue.

Preferably, the lindera root extract has the ability to inhibit the uptake of cholesterol and ox-LDL by macrophages, reducing macrophage lipid deposition.

Preferably, the active ingredient of the medicament comprises an extract of lindera root.

Preferably, the medicament further comprises a pharmaceutically acceptable carrier.

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

The invention provides a lindera root extract for treating hyperlipidemia, a preparation method and application thereof. The invention adopts the modern pharmacological means, ELISA, RT-PCR/WB, fluorescent probe and labeling and other methods, and observes the regulating effect of the lindera extract on the RCT process through a hyperlipidemia-induced Hyperlipidemia (HLP) animal model, oxidized low density lipoprotein (ox-LDL) -induced foam cells and the like, and the research results show that the lindera extract prepared by the invention can reduce the serum TC, TG, LDL-C level of a hyperlipidemia mouse and improve the lipid deposition of liver tissues; the lindera root extract can effectively reduce serum cholesterol level, and the cholesterol reduction mechanism is related to up-regulating the expression level of liver tissues ABCA1, ABCG1, PPARalpha, LXRalpha and FXR related to the reverse cholesterol transport process, and promoting the conversion of cholesterol into bile acid for excretion; the Linderae radix extract has effects of inhibiting the uptake of cholesterol and ox-LDL by macrophages and reducing the lipid deposition of macrophages. The lindera root extract can endow the traditional qi-regulating medicine with new scientific connotation, and promote the development of new products of special blood lipid-lowering traditional Chinese medicines and the development and utilization of the lindera root.

Drawings

FIG. 1 is the effect of Linderae radix extract on RAW cell cholesterol esters; and (3) injection: ^△ p <0.05 or ^△△ p <0.01 compared to Control group; ^* p <0.05 or ^** p <0.01 compared to ox-ldl group;

FIG. 2 is the effect of Linderae radix extract on lipid deposition uptake by RAW cells; wherein A: control; b: ox-ld l; c: ox-ldl+WYH; d: ox-ldl+WYM; e: ox-ldl+WYL; and (3) injection: ^* p <0.05 or ^** p <0.01 compared to ox-ldl group;

FIG. 3 is the effect of Linderae radix extract on the ability of RAW cells to uptake ox-LDL;

FIG. 4 is the effect of Linderae radix extract on the ability of RAW cells to uptake ox-LDL; and (3) injection: ^* p <0.05 or ^** p <0.01 compared to the DiL-Ox-ldl group;

FIG. 5 shows the effect of Linderae radix extract on miR-33 high-expression RAW cell ABCA1/ABCG1/LXRa/SR-BI/PPARα gene expression; and (3) injection: ^△ p <0.05 or ^△△ p <0.01 compared to mimic NC groups; ^* p <0.05 or ^** p <0.01 compared to mimic groups

FIG. 6 shows the effect of Linderae radix extract on miR-33 low-expression RAW cell ABCA1/ABCG1/LXRa/SR-BI/PPARα gene expression; and (3) injection: ^△ p <0.05 or ^△△ p <0.01 compared to the Inhibit NC group; ^* p <0.05 or ^** p <0.01 compared to the Inhibit group;

FIG. 7 is a fully automated protein expression analysis system;

FIG. 8 is the effect of Linderae radix extract on the expression of ABCA1/ABCG1/SR-BI protein in high/low RAW expressing cells of miR-33; wherein A: miR-33 high expression RAW 264.7 cell ABCA1, ABCG1 and SR-BI protein expression strip chart; b: miR-33 low expression RAW 264.7 cell ABCA1, ABCG1 and SR-BI protein expression strip chart; c: ABCA1/GAPDH (mimic transfection set); d: ABCG1/GAPDH (mimic transfection set); e: SR-BI/GAPDH (mimic transfection set); f: ABCA1/GAPDH (inhibit transfection group); g: ABCG1/GAPDH (inhibit transfection group); h: SR-BI/GAPDH (inhibit transfection group); and (3) injection: ^△ p <0.05 or ^△△ p <0.01 compared to the mic/Inhibit NC group; ^* p <0.05 or ^** p <0.01 compared to the mic/Inhibit group;

FIG. 9 is the effect of Linderae radix extract on miR-33 high-expression RAW cell LXRa protein expression; and (3) injection: ^△ p <0.05 or ^△△ p <0.01 compared to the mic NC group; ^* p <0.05 or ^** p <0.01 compared to the mic group;

FIG. 10 is the effect of Linderae radix extract on miR-33 low-expressing RAW cell LXRa protein expression; and (3) injection: ^△ p <0.05 or ^△△ p <0.01 compared to the Inhibit NC group; ^* p <0.05 or ^** p <0.01 compared to the Inhibit group;

FIG. 11 is a graph showing the effect of Linderae radix extract on the fluorescence expression intensity of ox-ldl induced RAW cells ABCA1/ABCG 1/LXRa;

FIG. 12 shows the strong effect of Linderae radix extract on ox-ldl induced fluorescence expression of RAW cells ABCA1/ABCG 1/LXRa; and (3) injection: ^△ p <0.05 or ^△△ p <0.01 compared to Control group; ^* p <0.05 or ^** p <0.01 compared to Ox-ldl group;

FIG. 13 shows the effect of Linderae radix extract on fluorescence expression intensity of miR-33 high-expression RAW cells ABCA1/ABCG 1/LXRa;

FIG. 14 shows the effect of Linderae radix extract on fluorescence expression of miR-33 high-expression RAW cells ABCA1/ABCG 1/LXRa; and (3) injection: ^△ p <0.05 or ^△△ p <0.01 compared to the mic NC group; ^* p <0.05 or ^** p <0.01 compared to the mic group;

FIG. 15 shows the effect of Linderae radix extract on fluorescence expression intensity of miR-33 low-expression RAW cells ABCA1/ABCG 1/LXRa;

FIG. 16 is the effect of Linderae radix extract on fluorescence expression of miR-33 low-expression RAW cells ABCA1/ABCG 1/LXRa; and (3) injection: ^△ p <0.05 or ^△△ p <0.01 compared to the Inhibit NC group; ^* p <0.05 or ^** p <0.01 compared to the Inhibit group;

FIG. 17 is a graph showing the effect of lindera root extract on lipid index in mice with hyperlipidemia model; and (3) injection: ^△ p <0.05 or ^△△ p <0.01 compared to NC group; ^* p <0.05 or ^** p <0.01 compared to MC group;

FIG. 18 is the effect of Linderae radix extract on liver histopathology in mice (HE staining, 200X);

FIG. 19 is the effect of Linderae radix extract on indirect fluorescence staining of mouse liver SCA1 (200X);

FIG. 20 is the effect of Linderae radix extract on the expression of ABCA1/ABCG1/FXR/LXRa/PPARα gene during RCT in mice; and (3) injection: ^△ p <0.05 or ^△△ p <0.01 compared to NC group; ^* p <0.05 or ^** p <0.01 compared to MC group;

FIG. 21 is the effect of Linderae radix extract on the expression of ABCA1/ABCG1/FXR/LXRa/PPARα protein during RCT in mice; and (3) injection: ^△ p <0.05 or ^△△ p <0.01 compared to NC group; ^* p <0.05 or ^** p <0.01 compared to MC group;

FIG. 22 is a transcriptome experimental flow diagram;

FIG. 23 is a graph of differential mRNA principal component analysis;

FIG. 24 is a volcanic diagram of mRNA expression;

FIG. 25 is a cluster heat map of significantly different mRNAs;

FIG. 26 is a bar graph of the difference gene GO enrichment between the normal and model groups;

FIG. 27 is a bar graph of the model and lindera panel differential gene GO enrichment;

FIG. 28 is a normal and model set differential gene KEGG enrichment scatter plot;

FIG. 29 is a graph of a model and lindera panel differential gene KEGG enrichment scatter.

Detailed Description

The mass ratio of the combined spicebush root to the water is preferably 1:9-11, more preferably 1:10; the soaking time is preferably 25-30 min, more preferably 30min; the time for the condensation reflux is preferably 0.5 to 1.5 hours, more preferably 1 hour.

The method for obtaining the liquid medicine according to the invention further preferably comprises the following steps: mixing the residues with water, heating and boiling, condensing and refluxing for 40-50 min, and combining the liquid medicine; the mass ratio of the dregs to the water is 1:7-9; more preferably, the condensation reflux time is 45min, and the mass ratio of the dregs to the water is 1:8.

The lindera root extract can reduce the serum total cholesterol, triglyceride and low-density lipoprotein cholesterol level.

The lindera root extract can up-regulate the expression level of ABCA1, ABCG1, PPARα, LXRα and FXR in liver tissues.

The lindera root extract has the capability of inhibiting the ingestion of cholesterol and ox-LDL by macrophages, and reduces the lipid deposition of the macrophages.

The active ingredients of the medicament of the invention preferably comprise lindera root extract; the medicament also preferably includes a pharmaceutically acceptable carrier. The active ingredient in the medicine can take the lindera root extract as the only active ingredient, and the lindera root extract can also be combined with other active ingredients with the function of treating hyperlipidemia. The medicine of the invention comprises but is not limited to injection preparations, emulsion, ointment, granules, powder and oral liquid. The medicament of the invention also comprises a pharmaceutically acceptable carrier. The invention has no special limitation on other auxiliary materials contained in the medicine, and the auxiliary materials commonly used in the field of medicine can be adopted.

The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. In the following examples, conventional methods are used unless otherwise specified.

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

In the following examples, SPSS15.0 software was used for statistics and analysis, each group of data was represented by mean.+ -. Standard deviation (x-SD), and the differences between groups were t-tested for mean comparison of the two samples; the multiple sets of comparisons employ one-way analysis of variance. P <0.05 is statistically significant.

Example 1

Preparation of lindera root extract

The radix Linderae is provided by Tiantaishan radix Linderae Limited company (lot number 20211015) of Zhejiang red stone beam group, the raw materials are added with 10 times of water, soaked for 30min, heated and boiled, condensed and refluxed for 1h, filtered, and the liquid medicine is collected. Adding water into the residues, refluxing for 45min under 8 times twice, filtering, collecting the combined medicinal liquid, steaming, and lyophilizing to obtain radix Linderae lyophilized powder.

Example 2

Cell experiment

1. Experimental medicine

Taking 0.043g of freeze-dried powder prepared in the example 1, adding 200 mu L of DMSO, shaking for dissolution, adding a serum-free DMEM medium, and filtering with a 0.22 mu m filter head to finally obtain a freeze-dried powder mother solution prepared to be 2.5 g/mL. In the experiment, cells were dosed at different concentrations by dilution.

2 Experimental methods and results

2.1 Cell culture and passage

Normal growth RAW264.7 (Saibutu Biotechnology Co., ltd.) was cultured in DMEM medium (complete medium) containing 10% fetal bovine serum, placed in a 37℃incubator with 5% CO ₂, changed every 2 days, and passaged for the third day. Cell passage specific procedure: the grown cells were gently scraped off with a spatula and the cell suspension was pipetted onto a 10mL centrifuge tube. Centrifuging at 1200rpm for 5min, removing supernatant, adding complete culture medium to obtain cell suspension, removing 1/3 part of cell suspension, spreading into culture flask of 25cm ², and culturing in 5% CO ₂ incubator at 37deg.C.

2.2 Effect of Linderae radix extract on cell Activity

Cell suspensions of RAW264.7 in logarithmic growth phase were inoculated into 96-well plates at 100. Mu.L per well, and cultured in a 5% CO ₂ incubator at 37 ℃. After the cells were completely adherent, the cells were divided into 7 groups for administration, each control group (DMEM medium), and each group was provided with 6 duplicate wells, each group containing 1250.0. Mu.g/mL, 625.0. Mu.g/mL, 312.5. Mu.g/mL, 156.3. Mu.g/mL, 78.1. Mu.g/mL, and 39.1. Mu.g/mL of the lindera root extract. After 24, 48h of corresponding culture intervention for each group, cell morphology was observed and activity was detected with CCK8 kit. After adding 10. Mu.L of CCK8 reagent per well and incubating in an incubator for 1 hour, the absorbance (OD) of each well was measured at 450nm in an ELISA reader, and the cell activity was calculated. Cell proliferation rate= (experimental group-control group)/control group×100%.

The effect of different concentrations and different times of lindera root extract (WY) on RAW264.7 cell proliferation was examined. The specific results are shown in Table 1.

Table 1 Effect of Lindera root extract on RAW cell proliferation Activity

Note that: p <0.05 or p <0.01 compared to Control group;

The results in Table 1 show that WY 39.1-156.3 μg/mL has no significant effect on RAW cell proliferation (p > 0.05) after 24 and 48 hours of intervention compared to the control group. WY 312.5-1250.0 mu g/mL is used for intervening 24-48 hours, and RAW cell proliferation is obviously inhibited (p is less than 0.01); and the inhibition rate reaches 54.84% after 1250.0 mug/mL is intervened for 24 hours, and the inhibition rate reaches 88.47% after 48 hours of intervention. Suggesting that the radix linderae extract has no obvious effect on RAW cell proliferation in the dosage range of less than 156.3 mug/mL; the radix linderae extract is more than 312.5 mug/mL to obviously inhibit RAW cell growth; and after 48 hours, the intervention effect is more obvious. Three doses of 156, 78, 39 μg/mL were then selected for subsequent experiments.

2.3 Effect of Lindera root extract on ox-LDL induced foam macrophage cholesterol ester content

RAW264.7 cells were seeded in 24-well plates and randomly divided into a control group (RAW 264.7 cells in normal culture), a foam cell group (RAW 264.7 cells in 100. Mu.g/mL ox-LDL treatment), a high-dose group of the extract of Linderae (RAW 264.7 cells in 100. Mu.g/mL ox-LDL+156. Mu.g/mL treatment), a dose group of the extract of Linderae (RAW 264.7 cells in 100. Mu.g/mL ox-LDL+78. Mu.g/mL treatment), a low-dose group of the extract of Linderae (RAW 264.7 cells in 100. Mu.g/mL ox-LDL+39.0. Mu.g/mL treatment) and 24 hours each group was provided with 3 wells. After 24h of drug intervention, the cells were scraped and collected, centrifuged at 1200rpm for 5min, the supernatant was discarded, 80 μl of absolute ethanol was added for ultrasonic extraction of total cholesterol, 10000r·min ^-1 was centrifuged for 5min, the supernatant was removed, and the Total Cholesterol (TC) and Free Cholesterol (FC) contents were determined according to the lipid assay kit instructions, respectively, cholesterol Ester (CE) content = TC-FC.

After 24h incubation of the lindera extract with ox-LDL, the effect of lindera on cholesterol esters of RAW cells was examined. The results in FIG. 1 show that the ox-LDL model group had significantly more cholesterol esters (p < 0.01) than the control group; the lindera extract has significantly reduced cholesterol ester content (p < 0.05) at high, medium and low doses compared to ox-LDL model group. It is suggested that lindera root extract has the ability to inhibit cholesterol uptake by macrophages.

2.4 Effect of Lindera root extract on ox-LDL induced foam macrophage lipid deposition

RAW264.7 cells were seeded in 24-well plates and randomly divided into a control group (RAW 264.7 cells in normal culture), a foam cell group (RAW 264.7 cells in 100. Mu.g/mL ox-LDL treatment), a high-dose group of the extract of Linderae (RAW 264.7 cells in 100. Mu.g/mL ox-LDL+156. Mu.g/mL treatment), a dose group of the extract of Linderae (RAW 264.7 cells in 100. Mu.g/mL ox-LDL+78. Mu.g/mL treatment), a low-dose group of the extract of Linderae (RAW 264.7 cells in 100. Mu.g/mL ox-LDL+39.0. Mu.g/mL treatment) and 24 hours each group was provided with 3 wells. Oil red O staining is carried out after the drug intervention for 24 hours, and the specific steps are as follows:

After the medicine is dried, the supernatant is discarded, the medicine is washed 3 times with PBS, 4% paraformaldehyde is fixed for 10min, PBS is washed 1 time, 60% isopropanol is fixed for 5min, 0.5% oil red O is added after the isopropanol is absorbed, the medicine is dyed for 20min at 37 ℃, the supernatant is discarded, PBS is washed 3 times, and the medicine is observed under a microscope and photographed in time.

After 24h incubation of the lindera extract with ox-LDL, lipid deposition was observed by oil red O staining. FIG. 2 shows the results showing increased lipid deposition in the ox-LDL model group compared to the control group; the lindera extract showed significantly reduced lipid deposition at high, medium and low doses (p < 0.01) compared to ox-LDL model group. It is suggested that the lindera root extract has the effect of improving macrophage lipid deposition.

2.5 Effect of Linderae radix extract on the ability of macrophages to ingest ox-LDL

RAW264.7 cells were seeded in 24-well plates, 2.5X 10 ⁵/well, randomly divided into control group (RAW 264.7 cells in normal culture), foam cell group (RAW 264.7 cells in normal culture for 24 hours in DiI-ox-LDL treatment), high dose group of Linderae extract (RAW 264.7 cells in DiI-ox-LDL+156. Mu.g/mL treatment for 24 hours in RAW264.7 cells in 100. Mu.g/mL DiI-ox-LDL+78. Mu.g/mL treatment for RAW264.7 cells in 24 hours in RAW extract), low dose group of Linderae extract (RAW 264.7 cells in 100. Mu.g/mL DiI-ox-LDL+39.0. Mu.g/mL treatment for 24 hours in RAW264.7 cells in RAW). After 24h of drug intervention, the high content cell imaging system performs fluorescence shooting and counts the fluorescence intensity. The specific results are shown in FIGS. 3-4 and Table 2.

Table 2 influence of Linderae radix extract on the ability of RAW cells to uptake ox-LDL (n=5)

Note that: p <0.05 or p <0.01 compared to DiL-Ox-ldl group.

After 24h incubation of the lindera extract with Dil ox-LDL, the results of FIGS. 3,4 and Table 2 show that the high, medium and low dose fluorescence expression of the lindera extract is significantly reduced (p <0.01, 0.05) compared to the control group. It is suggested that lindera root extract has the ability to inhibit the uptake of ox-LDL by macrophages.

2.6 Preparation of miR-33 high-expression cell model (taking a 6-orifice plate-12 mL system as an example)

Transfection reagent A solution: 660 mu L serum-free DMEM+42 mu LTRANSMANTE, and standing for 5min

Transfection reagent B solution: 695 μL serum free DMEM+7μ LmiR-33 (inhibit/mimic), and allowed to stand for 5min. And adding the solution B into the solution A, uniformly mixing, and standing at room temperature for 20min to obtain a mixed solution C. The old medium in the 6-well plate was discarded, 1.8mL of DMEM medium containing 10% FBS was added to each well, and 200. Mu.L of the mixture C was added to each well. After culturing for 24 hours in an incubator, discarding the culture solution, collecting RNA, and detecting the miR-33 expression condition by RT-PCR.

2.7RT-PCR detection of influence of Linderae radix extract on miR-33 high/low expression cells ABCA1, ABCG1, PPARα, LXRα, SR-BI and other gene expression

RAW264.7 cells in the logarithmic growth phase are evenly plated in a six-hole plate, placed in a cell incubator with 5% CO ₂ at 37 ℃ for continuous culture, and subjected to grouping and molding according to 2.6 items when the cell confluence is 80%. And after the lindera root extracts with different concentrations interfere with miR-33 high/low expression RAW264.7 cells for 24 hours, extracting total RNA by using a cell RNA rapid extraction kit. Specifically, 500uL of lysate is added into each 6-well plate, and the mixture is vibrated for 1min at room temperature. The lysate was collected and added to a DNA removal column, centrifuged at 12,000rpm for 60s, and the filtrate was collected. 700. Mu.L of deproteinized solution RW1 was centrifuged at 12,000rpm for 60s at room temperature of 1min, and the filtrate was discarded. 500. Mu.L of rinsing liquid RW was added to the column, centrifuged at 12,000rpm for 60s, and the waste liquid was discarded and repeated 1 time. 13,400 Xg 2min, 30. Mu. L RNASE FREE H ₂ O was added, centrifuged at 12,000rpm for 60s, and the liquid was collected and stored at-80 ℃.

Reverse transcribing the extracted RNA into cDNA according to the instruction of the kit, uniformly mixing the RNA with gDNA, removing mixed solution and ddH ₂ O without RNase, incubating for 2min at 42 ℃, adding stem-loop primer, RT premix and reverse transcriptase, and performing reverse transcription reaction in a PCR instrument according to the following reaction conditions: 25 ℃ for 5min,50 ℃ for 15min and 85 ℃ for 5min. After completion of the reverse transcription, primers (shown in Table 3) were designed and the expression of ABCA1, ABCG1, PPARα, LXRα, SR-BI and miRNA-33 was detected by RT-PCR. The specific results are shown in FIGS. 5-6 and tables 4-5.

FIG. 5-FIG. 6 and Table 4-Table 5 mimic are examples of induction of high gene expression; mimic NC is non-intervention mimic; the inhibit is the low expression of the induced gene; an inhibit is an uninterfered inhibit.

TABLE 3 Gene sequence

Table 4 Effect of Lindera root extract on miR-33 high-expression cell Gene expression

Note that: ^△ p <0.05 or ^△△ p <0.01 compared to mimic NC groups; ^* p <0.05 or ^** p <0.01 compared to mimic groups.

Influence of Lindera root extract on miR-33 low-expression cell gene expression in Table 5

Note that: ^△ p <0.05 or ^△△ p <0.01 compared to InhibitNC groups; ^* p <0.05 or ^** p <0.01 compared to the Inhibit group.

Compared with mimic NC groups, the gene expression of miR-33 high-expression cells ABCA1, ABCG1, LXRA and SR-BI is significantly reduced (p is less than 0.01), and PPARα has no significant change. Compared with the miR-33mimic transfected group, the combined spicebush root high dose can obviously increase the expression level (p < 0.05) of ABCA1, LXRA and SR-BI genes in miR-33 high-expression cells; the dosage of the combined spicebush root can obviously increase the SR-BI gene expression level (p < 0.01) in miR-33 high-expression cells; the low dosage of the combined spicebush root obviously reduces the expression level (p <0.01, 0.05) of ABCA1, ABCG1, LXRA and SR-BI genes in miR-33 high-expression cells.

Compared with inhibitNC groups, the gene expression of miR-33 low-expression cells ABCA1, ABCG1, LXRA, SR-BI and PPARα is significantly up-regulated (p < 0.01). Compared with miR-33inhibit transfection group, the combined spicebush root high and medium doses can obviously increase the expression level (p <0.01, 0.05) of ABCG1, LXRA, SR-BI and PPARalpha genes in miR-33 low-expression cells, and have obvious dose dependence; the low dosage of the combined spicebush root obviously reduces the expression level (p <0.01, 0.05) of the ABCA1, ABCG1 and LXRa genes in miR-33 low-expression cells.

2.8Wes full-automatic protein expression analysis System for detecting influence of Linderae radix extract on miR-33 high/low expression cells ABCA1, ABCG1 and SR-BI protein expression

After dry pre-transfection of RAW264.7 cells of miR-33 with the lindera root extract for 24 hours, the cells were collected with a scraper, centrifuged at 1250rpm for 5min, the supernatant was discarded, washed with PBS and centrifuged again. Then 100. Mu.L of RIPA lysate containing protease and phosphatase inhibitor was added, and the extraction was performed in ice box for 1min with ultrasound, and for 5s every 30 s. Then, the cell lysate is put into a refrigerator at 4 ℃ and is uniformly mixed every 10min, so as to ensure full lysis. After 40min, the supernatant was collected by centrifugation at 12000rpm for 10min, and the BCA was used for detecting the cell concentration. And detecting the expression of the corresponding protein by adopting Wes full-automatic protein expression analysis system. The specific results are shown in FIG. 8.

As can be seen from FIG. 8, the miR-33 highly-expressed cells ABCG1, SR-BI and ABCA1 protein expression is significantly reduced (p < 0.01) compared with mimic NC groups. Compared with miR-33mimic transfected group, the combined spicebush root high dose can significantly increase the expression of ABCG1 and ABCA1 proteins (p < 0.01); the dosage of the combined spicebush root can obviously increase the SR-BI and ABCA1 protein expression level (p < 0.01) in miR-33 high-expression cells; the low dosage of the combined spicebush root has obvious effect on increasing the expression of ABCA1 (p < 0.01) in miR-33 high-expression cells and has no obvious effect on the expression level of ABCG1 and SR-BI protein. Compared with inhibitNC groups, miR-33 low-expression cell ABCG1 protein expression is significantly up-regulated (p < 0.05). Compared with miR-33inhibit transfection group, the combined spicebush root high and medium doses can obviously raise ABCG1 (p <0.05,0.01) in miR-33 low-expression cells, and has a slight raising trend on SR-BI protein expression, but no obvious difference. The low dosage of the combined spicebush root has no obvious influence on the expression level of the ABCG1, SR-BI and ABCA1 proteins in miR-33 low-expression cells.

2.9Westernblot detection of LXR alpha protein expression in cells after miR-33 transfection

After dry pre-transfection of RAW264.7 cells of miR-33 with the lindera root extract for 24 hours, the cells were collected with a scraper, centrifuged at 1250rpm for 5min, the supernatant was discarded, washed with PBS and centrifuged again. Then 100. Mu.L of RIPA lysate containing protease and phosphatase inhibitor was added, and the extraction was performed in ice box for 1min with ultrasound, and for 5s every 30 s. Then, the cell lysate is put into a refrigerator at 4 ℃ and is uniformly mixed every 10min, so as to ensure full lysis. After 40min, the supernatant was collected by centrifugation at 12000rpm for 10min, and the BCA was used to measure the cell concentration, and the loading amount was 20. Mu.g. And detecting LXR alpha protein expression by adopting Westernblot after sample denaturation. The specific results are shown in fig. 9 and 10.

Compared to mimic NC groups, miR-33 high-expression cells LXRa expression was significantly down-regulated (p < 0.01). Compared with miR-33mimic group, the combined spicebush root can be obviously increased by LXRa (p < 0.01) at a high dose, and the result is shown in figure 9. Compared with inhibitNC groups, miR-33 low-expression cells LXRa protein expression is not significantly up-regulated. Compared with the miR-33inhibit transfection group, the combined spicebush root high and medium doses can increase the expression of LXR-a in miR-33 low-expression cells, but no significant difference exists, and the result is shown in figure 10.

2.10TSA detection of Effect of Linderae radix extract on ox-ldl Induction, miR-33 high/low expression cells ABCA1, ABCG1 and LXR alpha protein fluorescence expression

Taking cells in logarithmic growth phase, uniformly plating the cells in a six-hole plate, placing the cells in a cell incubator with 5% CO ₂ at 37 ℃ for continuous culture until the cell confluence is 80%, and grouping and molding. After the lindera root extracts with different concentrations intervene miR-33 high-expression RAW cells for 48 hours, the supernatant is poured, PBS is used for washing 3min/3 times, 4% paraformaldehyde is fixed for 20min, and PBS is used for washing 3min/3 times. Adding 0.5% TritonX-100 (PBS) and washing for 3min/3 times at room temperature, absorbing the blocking liquid by using water absorbing paper, not washing, adding ABCA1, ABCG1 and LXR alpha antibody (1:200), incubating overnight at 4 ℃, washing for 3min/3 times by using PBS, absorbing excessive liquid by using water absorbing paper, then dripping corresponding secondary antibody, incubating for 1h at room temperature, washing for 3min/3 times by using PBS, developing DAB, washing for 3min/3 times by using PBS, carrying out hematoxylin nuclear staining, washing off floating color, airing and photographing. The specific results are shown in FIGS. 11-16.

As can be seen from fig. 11 and 12, there was no significant change in ABCA1 expression (p > 0.05), significantly reduced ABCG1 expression (p < 0.01), and significantly increased LXRa expression (p < 0.01) after Ox-ldl molding compared to the Control group. Compared to Ox-ldl model group, the high dose of combined spicebush root significantly increased ABCG1 expression (p < 0.01), the dose of combined spicebush root significantly increased ABCA1 and LXRa expression (p < 0.01), and the low dose of combined spicebush root significantly increased LXRa protein expression (p < 0.01).

From fig. 13 and 14, miR-33 highly-expressing cells ABCA1, ABCG1 expression were significantly down-regulated (p < 0.01), and LXRa expression was significantly up-regulated (p < 0.01) compared to mimic NC groups. Compared with the miR-33mimic transfected group, the combined spicebush root high dose can obviously increase the expression level (p < 0.01) of ABCA1 and LXRa proteins in miR-33 high-expression cells; the dosage in the combined spicebush root can obviously increase the expression level (p < 0.01) of ABCA1, ABCG1 and LXRa proteins in miR-33 high-expression cells; the low dosage of the combined spicebush root obviously increases the expression level (p <0.01, 0.05) of the ABCA1, ABCG1 and LXRa proteins in miR-33 high-expression cells.

From fig. 15 and 16, protein expression was significantly up-regulated in miR-33 hypoexpressing cells ABCA1, LXRa (p < 0.01) compared to inhibitNC groups. Compared with miR-33inhibit transfection group, the combined spicebush root high dose can obviously increase the expression level (p < 0.01) of ABCA1 and LXRa genes in miR-33 low-expression cells; the expression level of LXRa genes in miR-33 low-expression cells is obviously increased in the medium and low doses of the combined spicebush root (p < 0.01).

Example 3

Animal experiment

1 Material

Male C57BL/6J mice, 36, weighing 20+ -2 g, were purchased from Hangzhou open-Authentic laboratory animal technologies Inc. (animal quality certificate number: 20230117Abzz0100999730, license number: SCXK (Zhe) 2022-0005). Raising at room temperature of 23+/-2 ℃ and relative humidity of 40% -70%, and drinking water by free diet. Animal ethical lot number: 20220909.

The freeze-dried powder prepared in example 1 was prepared by preparing 200 mg/mL ^-1 of suspension with pure water. Positive control: atorvastatin calcium tablet (trade name Lipitor, pyroxene pharmaceutical Co., ltd.) was prepared as a suspension at a concentration of 0.15 mg/mL ^-1 with distilled water immediately before use.

2 Experimental methods and results

2.1 Model replication and group administration

Mice were divided into 6 groups of a normal control group, a hyperlipidemia model control group, an atorvastatin control group (6.0 mg. Kg ^-1), a miR-33agomir combined spicebush intervention group (2.0 g. Kg ^-1), a miR-33antagomir combined spicebush intervention group (2.0 g. Kg ^-1) and a combined spicebush intervention group (2.0 g. Kg ^-1), each group being 6. Normal groups were given normal feed and water, the remaining groups were given high fat feed for 4 weeks continuously, and a hyperlipidemic model was replicated; the miR-33mimics combined spicebush root group and the miR-33inhibitor combined spicebush root group are respectively injected into the abdominal cavity by 10 mg.kg ^-1 miR-33agomir and 10 mg.kg ^-1 miR-33antagomir, and physiological saline is injected into the abdominal cavity of the other mice once a week for 4 weeks. The first week was followed by a boost 2 days after the first injection, with the same dose. Atorvastatin is prepared with purified water at a concentration of 0.15 mg.mL ^-1 and Wu Yaoshui extract at a concentration of 200 mg.mL ^-1, and the intragastric volume is 1mL 100g ^-1 body mass, administered once daily, beginning with the corresponding drug intervention at week 5 for 4 consecutive weeks.

2.2 Effect of Wu Yaoshui extracts on HLP model mice blood lipid

The method comprises the steps of collecting blood from the eyesockets of the mice, centrifuging, separating serum, killing the mice by a neck removing method, separating livers, cleaning blood stains by using ice physiological saline, sucking up by using filter paper, immediately freezing a part of the blood stains in liquid nitrogen for standby, and placing a part of the blood stains in a 4% paraformaldehyde solution for standby after shearing a proper size. Full-automatic biochemical tests detect TC, TG, HDL-C, LDL-C levels in each sample. The specific results are shown in FIG. 17.

As can be seen from FIG. 17, the serum TC, TG, LDL-C levels of mice in the model group were significantly increased (P < 0.05, P < 0.01) compared to the corresponding index in the normal group, and the HDL-C levels were significantly decreased (P < 0.05) compared to the normal group; compared with the model group, the black-bone liquid extract can significantly reduce the serum TC, TG and LDL-C levels (P < 0.01) of mice.

2.3 Effect of Linderae radix extract on liver histopathology in mice

Hematoxylin-eosin staining (HE) mice were observed for liver histopathological changes. The liver tissue of the mouse is taken out from the 4% paraformaldehyde solution after fixation, dehydrated by alcohol, and the xylene is transparent after dehydration. And then carrying out wax dipping and embedding, slicing the slices with the thickness of 5um by using a paraffin slicer, and drying the slices on a slice dryer for 2 hours. Paraffin was washed off and xylene was washed off with 100% alcohol for 5 min. Soaking in alcohol, dyeing with hematoxylin solution for 30min, washing off the floating color with tap water, and counterstaining with eosin solution for 1-10min. And (3) placing the dried glass into xylene for transparency, dripping neutral gum on a glass slide, covering and sealing the glass slide, observing the dyeing condition under an optical microscope, and taking a picture. The specific results are shown in FIG. 18.

As can be seen from FIG. 18, a small amount of glycogen accumulates in the normal group. The model group can see fatty vacuoles, basophils, or extramedullary hematopoiesis, or inflammatory leukocyte foci. Atorvastatin group had reduced fat vacuoles and fibrosis. The miR33ago +combined spicebush root group has a plurality of basophilic staining cell foci, and lipid vacuoles are not obviously reduced. The miR33ant + combined spicebush root group can see a reduction of lipid vacuoles and basophils foci. The lindera aggregate lipid vacuoles and basophils foci are reduced.

2.4 Effect of Linderae radix extract on Indirect fluorescent staining of mouse liver SCA1

Liver sections were dewaxed and rehydrated, washed three times with PBS for 5min each. The sodium citrate repairing liquid is subjected to microwave repairing, medium fire for 7min and low fire for 10min. After cooling to room temperature, the cells were washed three times with PBS. The 3% hydrogen peroxide was blocked for 15min and washed three times with PBS. Immunostaining was performed rapidly with the blocking fluid blocked for 15min. The cells were washed three times with PBS, and then were incubated overnight at 4 ℃. The solution was washed three times with PBS and incubated for 30min at 37 ℃. The PBS was washed three times again. DAPI-containing anti-fluorescence quenching caplets were added dropwise. Shooting under a fluorescence microscope. The specific results are shown in FIG. 19.

The results in FIG. 19 show that the model group has a higher fluorescence intensity than the normal group, indicating that the extramedullary hematopoiesis is more active and that the fat content of the model group is increased; compared with the model group, the combined spicebush root group has reduced fluorescence intensity, which shows that the extramedullary hematopoiesis is less and fat deposition is improved. Blue fluorescent plaques are some of the aggregation of primitive blood cells.

2.5 Effect of Linderae radix extract on the levels of transcription factors and protein mRNA related to the mouse RCT Process

Detecting expression levels of liver tissues miR-33, ABCA1, ABCG1, PPARα, LXRα and FXRmRNA by adopting RT-PCR: extracting total RNA by using a Trizol kit, synthesizing cDNA by reverse transcription according to the kit specification, detecting the relative expression levels of target genes in different samples by using RT-PCR, recording Ct values of the target genes and internal reference genes, and calculating Ct difference and expression levels of the target genes and the internal reference genes. Primer design (see Table 6) and order from Shanghai, inc. of biological engineering (Shanghai). The specific results are shown in FIG. 20.

TABLE 6 Gene sequence

The results of FIG. 20 show that the expression of ABCA1, ABCG1 and miR-33mRNA in liver tissues of mice in the MC group has an increasing trend, the expression of FXR and PPARα mRNA has an insignificant change, and the expression of LXRa mRNA has a decreasing trend, compared with the NC group; compared with MC group, radix Linderae can raise the expression level of ABCA1, ABCG1, FXR, LXRA and PPARα mRNA of mouse liver tissue, and reduce the expression level of miR-33 mRNA. mRNA changes were not evident in the miR33ago +WY group and the miR33ant+WY group.

2.6 Effect of Linderae radix extract on expression of transcription factor protein related to mouse RCT process

Respectively taking a small amount of liver tissue, fully grinding in a liquid nitrogen environment, carrying out ice lysis on RIPI lysate, measuring the protein concentration by a BCA method, adding an equal volume of 2 Xloading buffer solution, and carrying out denaturation at 100 ℃ for 10min. Equal amounts of total protein were taken from each sample and analyzed by 20% SDS-PAGE electrophoresis: preparing 8-12% separating gel and 5% concentrating gel, loading 60 μg total protein in each well, 10-15 μl in each well, concentrating gel 60V, and electrophoresis at 80V for about 2 hr. PVDF film transfer: soaking PVDF membrane in methanol for 20sec, transferring to Tris-Glycine transfer buffer (containing 5% methanol) and balancing for at least 5min; SDS-PAGE gels were equilibrated in Tris-Glycine transfer buffer for at least 30min; full wet transfer was performed at a constant pressure of 100v for 2h under cooling. After the transfer was completed, the membrane was put into T-TBS (containing 5% BSA)), blocked at room temperature for 1 hour, and then rinsed with T-TBS for 5 min. Times.3. Primary antibodies (ABCA 1, ABCG1, pparα, lxrα, FXR) were incubated overnight at 4 ℃, HRP-labeled secondary antibodies were incubated for 2h at room temperature usingWest Dura Extended Duration Substrate, operating according to the instruction, preparing about 1ml of ECL working solution, incubating the transfer film for 1min at room temperature, removing redundant ECL reagent, sealing the preservative film, placing the X-ray film in the cassette for exposure for 5-10min, and then developing and fixing. The optical density values of the bands were analyzed using Image J software, each band was repeated 3 times, and the relative expression amount of the target protein = { target protein (optical density value)/internal reference (optical density value) } ×10n was expressed, and the results were expressed as mean ± standard deviation. The specific results are shown in FIG. 21.

The results in fig. 21 show that ABCA1, ABCG1, FXR, LXRa, PPAR a protein expression was significantly down-regulated in the MC group compared to the NC group (p < 0.01). Compared with MC group, radix Linderae can significantly increase ABCA1, ABCG1, FXR, LXRa, PPAR alpha protein expression (p < 0.01); ABCA1, ABCG1, FXR, LXRa, PPAR a protein expression in miR33ant + combined spicebush root group was significantly up-regulated (p < 0.01). The expression of ABCA1, ABCG1, FXR and LXRA proteins in the miR33ago +combined spicebush root group is obviously up-regulated (p <0.01, 0.05), and the expression level of PPARalpha proteins has no obvious effect.

2.7 Small mouse book acquisition and transcriptome sequencing

After the blood is collected from the eyebox of the mouse, the liver of the mouse is killed and separated by a cervical method, one part of the liver is immediately frozen in liquid nitrogen for standby, and the other part of the liver is sheared to a proper size and placed in 4% paraformaldehyde solution for standby. RNA from mouse liver was isolated and purified using TRIzol according to the protocol. Quality control of total RNA amount and purity was then performed using NanoDrop ND-1000, and RNA integrity was checked by Bioanalyzer 2100; concentration >50 ng/. Mu.L, RIN value >7.0, total RNA > 1. Mu.g satisfied the downstream experiments. Finally, it was double-ended sequenced using illuminaNovaseqTM 6000,6000 according to standard procedures, in the sequencing mode PE150. The transcriptome experimental flow is shown in FIG. 22.

2.8 Transcriptome sequencing mRNA data quality control

Raw data generated by sequencing is subjected to pretreatment and data filtering to obtain effective data (CLEAN DATA), and then the effective data is compared with a reference genome, and finally, quantitative analysis of genes is performed. The sample quality control statistics are shown in Table 7.

TABLE 7 sample quality control statistics

Note that: NC normal group; MOD model sets; WY lindera root group; miR33ant: miR33 ant+combined spicebush root group; miR33ago: miR33ago + Lindera root group.

2.9 Differential expression analysis

2.9.1 Differential Gene totality

The number of differentially expressed genes (|log2fc| > =1 & q < 0.05) compared between the two groups was counted and shown in table 8.

Table 8 differential expressed genes compared between two groups

2.9.2 Differential mRNA principal component analysis

The Principal component analysis (Principal ComponentAnalysis, PCA) can intuitively observe the sample clustering condition of each group, and the difference mRNA Principal component analysis chart is shown in FIG. 23.

The main principle of PCA is dimension reduction, and as a result, if samples are clustered together, it is indicated that the differences between the groups of samples are small; if the distance between samples is far, this means that the differences between the groups of samples are large. From the results shown in fig. 23, the differences among the three groups of the normal group, the model group and the combined spicebush root group are found to be significant, and the differences among the samples in the groups are small, which indicates that the sample repeatability is high.

2.9.3 Volcanic image of differential mRNA expression

Red represents up-regulation, blue represents down-regulation, and gray represents no significant difference, see fig. 24.

FIG. 24 shows that there are 1736 model group differential mRNAs, 1357 up-regulated genes and 379 down-regulated genes, compared to the normal control group; compared to the model group, there were 25 lindera panel differential mrnas, 12 up-regulated genes and 13 down-regulated genes. As a result of the significant difference mRNA heat pattern, the model group was compared with the normal group, and the difference mRNA was Cyp2a4, cyp2b9, slco a1, csad, fdps, C6, serpina, sqle, mup3, mvd, cyp51, etc., and the lindera group was compared with the model group, and the difference mRNA was Rdh f1, gm49380, guca1a, cyp2d12, igkv4-61, hcn3, etc. Wherein Cyp2a4 and Cyp2b9 are associated with steroid hydroxylase activity; slco1a1 is associated with bile acid and bile salt transport; csad is associated with the process of decomposition of l-cysteine to taurine; fdps, serpin a12, sqle, mvd, and Cyp51 are associated with lipid synthesis and metabolic processes; rdh16f1 is associated with oxidoreductase activity; gm49380 is associated with endoplasmic reticulum transferase activity; guca1a is associated with calcium sensitive guanylate cyclase activator activity.

2.9.4 Significant difference mRNA thermogram

Columns represent samples, rows represent genes, red represents high expression, and blue represents low expression. See fig. 25.

FIG. 25 shows that the differential mRNAs are Cyp2a4, cyp2b9, slco a1, csad, fdps, C6, serpina12, sqle, mup3, mvd, cyp51, etc. in the model group compared to the normal group, and Rdh f1, gm49380, guca1a, cyp2d12, igkv4-61, hcn3, etc. in the combined spicebush group compared to the model group.

2.10 Differential Gene GO enrichment Bar charts

The differential gene GO was enriched for the most significant 20 at the bar p-value level, see fig. 26 and 27.

In the result of GO enrichment analysis, the first 20 items related to biological processes of model mice mainly relate to immune system processes, bacterial reactions, inflammatory reactions, reactions to stilbene, innate immune reactions, steroid metabolic processes, cholesterol biosynthesis processes, cholesterol metabolic processes, steroid biosynthesis processes and the like. Thus, the onset of hyperlipidemia is predicted to be associated with lipid metabolism such as cholesterol biosynthesis and metabolic processes, steroid biosynthesis and metabolic processes, and biological processes such as inflammatory reactions and immune system processes.

The cell composition-related entries relate mainly to cell surface, plasma membrane outside, extracellular space, etc. Thus, it is predicted that the target site associated with hyperlipidemia is mainly located on the cell surface, outside the plasma membrane, or the like. The molecular function related entries relate mainly to small molecule binding, oxidoreductase activity, acting on the paired donor, binding or reduction, oxidoreductase activity, scavenger receptor activity, transmembrane signaling receptor activity, steroid hydroxylase activity. Thus, it is predicted that the onset of hyperlipidemia may be associated with molecular functions such as small molecule binding, oxidoreductase activity, cytokine receptor activity, scavenger receptor activity, and the like.

The first 20 items related to biological processes in the GO enrichment analysis result of the mice after the dry period of the combined spicebush root mainly relate to negative regulation of the quinolinate biosynthesis process, secondary metabolic processes, regulation of T cell steady-state proliferation, forward regulation of cGMP-mediated signals, retinoic acid metabolic processes and protein K11-linked ubiquitination. Thus, the predicted target of the combined spicebush root for treating the hyperlipidemia may be related to the biological processes such as negative regulation of the quinolinate biosynthesis process, regulation of T cell steady-state proliferation, ubiquitination and the like. The cell composition-related entries relate mainly to the hrs 1p ubiquitin ligase ERAD-L complex, signal recognition particles, outer nuclear membrane, components of the endoplasmic reticulum membrane, endoplasmic reticulum. Therefore, the predicted target of the combined spicebush root for treating the hyperlipidemia is mainly positioned at the positions of ubiquitin ligase, epicardium, endoplasmic reticulum membrane, endoplasmic reticulum, intracellular membrane combined with organelles and the like. The molecular function related entries relate primarily to aminocarboxylate-semialdehyde decarboxylase activity, interleukin-20 binding, guanylate cyclase modulator activity, and calcium-sensitive guanylate cyclase activator activity. Thus, the predicted target of the combined spicebush root for treating the hyperlipidemia may be related to the activity of the receptor ligand, the molecular functions of ubiquitin-specific protease and the like.

2.1 Differential Gene KEGG enrichment analysis

KEGG PATHWAY show the most significantly enriched 10 to determine the signal transduction pathways in which the differential genes between groups are primarily involved. See fig. 28 and 29.

In the KEGG pathway enrichment results, pathways with a significant anterior difference from the model group were found to have hematopoietic lineage, tuberculosis, osteoclast differentiation, cell adhesion molecules, steroid biosynthesis. The pathways leading to significant differences in the model group compared to the lindera aggregate group were steroid hormone biosynthesis, ascorbate and aldehyde acid salt metabolism, etc. Further verifies the research thought of the combined spicebush root for treating the hyperlipidemia by regulating the lipid synthesis process. Lays a solid foundation for the subsequent deep development of the mechanism of the combined spicebush root for resisting the hyperlipidemia and the related diseases of atherosclerosis.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. A method for preparing an lindera root extract, which is characterized by comprising the following steps: soaking radix Linderae in water, boiling, condensing, refluxing, and filtering to obtain medicinal liquid, namely radix Linderae extract; the time of condensation reflux is 0.5-1.5 h.

2. The preparation method according to claim 1, wherein the mass ratio of the combined spicebush root to the water is 1:9-11, and the soaking time is 25-30 min.

3. The method according to claim 1, wherein the obtained medicinal liquid further comprises: mixing the residues with water, heating and boiling, condensing and refluxing for 40-50 min, and combining the liquid medicine; the mass ratio of the dregs to the water is 1:7-9.

4. A lindera root extract obtained by the preparation method of any one of claims 1 to 3.

5. The use of the lindera root extract of claim 4 in preparing medicine for treating hyperlipidemia.

6. The use according to claim 5, wherein the lindera root extract is capable of lowering serum total cholesterol, triglyceride, low density lipoprotein cholesterol level.

7. The use according to claim 5, wherein the lindera extract is capable of up-regulating the expression levels of ABCA1, ABCG1, pparα, lxrα and FXR in liver tissue.

8. The use according to claim 7, wherein the lindera root extract has the ability to inhibit the uptake of cholesterol and ox-LDL by macrophages, reducing macrophage lipid deposition.

9. The use according to claim 5, wherein the active ingredient of the medicament comprises an extract of lindera root.

10. The use of claim 9, wherein the medicament further comprises a pharmaceutically acceptable carrier.