Application of dihydrobaicalin in the preparation of lipid-lowering drugs

Abstract

The present invention discloses the use of dihydrobaicalin in the preparation of lipid-lowering drugs. The present invention finds that dihydrobaicalin can effectively inhibit lipid accumulation in hepatocytes, and has the prospect of being developed into a lipid-lowering drug that inhibits lipid accumulation in hepatocytes. Those skilled in the art know that the difference between dihydrobaicalin and baicalin is only one double bond, and the activities of the two should be similar, but the effect of dihydrobaicalin in inhibiting lipid accumulation in primary liver cells is much stronger than that of baicalin, and this technical effect is unexpected by those skilled in the art.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and particularly relates to the application of dihydrobaicalin in the preparation of lipid-lowering drugs. Background Art

[0002] Nonalcoholic fatty liver disease is a metabolic disease with complex etiology, with ectopic hepatic lipid accumulation as the main pathological feature, including increased levels of hepatic free fatty acids, especially saturated fatty acids, which in turn induce fat toxicity. Excessive lipid accumulation may overwhelm the liver's ability to properly store or process fatty acids, leading to the production of toxic lipid substances, further inducing endoplasmic reticulum stress, oxidative stress and mitochondrial dysfunction in cells. Therefore, anti-hepatocyte lipid accumulation is the primary goal of treating nonalcoholic fatty liver disease. [Reference: Hepatokines: linking nonalcoholic fatty liver disease and insulin resistance. Nature reviews. Endocrinology, 2017]

[0003] The molecular formula of dihydrobaicalin (DBA) is C 21 H 20 O 11 , CAS number is 56226-98-3. There is no report that dihydrobaicalin has anti-hepatocyte lipid accumulation activity. Summary of the invention

[0004] The purpose of the present invention is to provide the use of dihydrobaicalin in the preparation of lipid-lowering drugs.

[0005] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0006] Application of dihydrobaicalin in the preparation of lipid-lowering drugs, wherein the lipid-lowering drug refers to the inhibition of lipid accumulation in hepatocytes.

[0007] Furthermore, the lipid-lowering drug uses dihydrobaicalin as an active ingredient and is prepared into a pharmaceutically acceptable dosage form using pharmaceutically acceptable excipients.

[0008] Furthermore, the auxiliary material is a solid, liquid or semi-solid auxiliary material.

[0009] Furthermore, the dosage forms include tablets, capsules and soft capsules.

[0010] Beneficial effects:

[0011] The present invention finds that dihydrobaicalin can effectively inhibit lipid accumulation in hepatocytes, and has the prospect of being developed into a lipid-lowering drug that inhibits lipid accumulation in hepatocytes. Those skilled in the art know that dihydrobaicalin and baicalin differ only in one double bond, and their activities should be similar, but the effect of dihydrobaicalin in inhibiting lipid accumulation in primary hepatocytes is much stronger than that of baicalin, and this technical effect is unexpected by those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The results of Bodipy staining in human liver cancer cells in each group;

[0013] Figure 2 The results of Nile red staining in human liver cancer cells in each group;

[0014] Figure 3 Bodipy staining results in primary liver cells of mice in each group;

[0015] Figure 4 The results of Nile red staining in primary liver cells of mice in each group; DETAILED DESCRIPTION

[0016] The essential contents of the present invention are described in detail below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto.

[0017] Example 1: Cell model based on human liver cancer cell HepG2 cells

[0018] 1. Experimental Materials

[0019] Dihydrobaicalin (DBA, Chengdu Pusi Biotechnology Co., Ltd., 56226-98-3);

[0020] human hepatoma cells (HepG2, National Cell Identification Library, Chinese Academy of Sciences (Shanghai, China);

[0021] Australian fetal bovine serum (FBS, Gibco, 10270-106);

[0022] Pancreatin (Gibco, 25200072);

[0023] DMEM high-glucose medium (containing double antibodies) (Keygene, KGM12800-500);

[0024] Bodipy 493 / 503 fluorescent probe (Thermo, D3922);

[0025] Nile red dye (Thermo, N1142);

[0026] Sodium hydroxide (NaOH, Sinopharm);

[0027] Bovine serum albumin (BSA, Bio-Tech ST023);

[0028] Palmitic Acid (Sigma, P0500).

[0029] 2. Experimental Methods

[0030] 1. Prepare palmitic acid (PA) solution: accurately weigh 25.642 mg PA, add 5 mL of 0.1 M NaOH, heat in a 70°C water bath until clear to fully saponify it, then add 5 mL of 10% BSA solution to cross-link it to prepare a 10 mM stock solution, and finally dilute it with culture medium to the required concentration for the experiment.

[0031] 2. Cell culture

[0032] HepG2 cells were cultured and passaged in DMEM high-glucose medium containing FBS (containing double antibodies) at 37°C and 5% CO2, and F5-F6 generation cells were used for the experiment.

[0033] 3. Detection of lipid accumulation in HepG2 cells by Bodipy staining

[0034] (1) HepG2 cells with good growth status were taken and resuspended in DMEM high-glucose medium containing 10% FBS, and inoculated into 96-well plates, with 4.5×10 cells per well. 4 / mL, divided into control group (Control), model group (PA), BA group (10μM) and DBA group (10μM), each group of 6 replicates. After 24h, the control group was replaced with fresh culture medium, the model group was replaced with fresh culture medium containing 200μmol / mLPA, the BA group was replaced with fresh culture medium containing 200μmol / mLPA and 10μM BA, and the DBA group was replaced with fresh culture medium containing 200μmol / mLPA and 10μM DBA.

[0035] (2) After culturing for 24 h, the original culture medium was discarded and 100 μL PBS was added to each well for rinsing twice.

[0036] (3) Prepare mixed staining solution I. Dilute the two dyes into PBS solution at a ratio of 1:10000 of Bodipy stock solution and 1:1000 of Hoechst solution. Vortex thoroughly to mix and store in a dark place.

[0037] (4) Add 100 μL of mixed staining solution I to each well and incubate at 37°C in the dark for 30 min.

[0038] (5) Aspirate and discard the mixed staining solution I, gently wash with PBS three times, add 100 μL PBS to each well, and then use a high-content imaging analyzer to collect lipid accumulation phenotype data.

[0039] (6) Image phenotypic parameters were collected based on a high-content imaging system. A 40× water objective was selected, and the confocal mode was used to collect 25 fields of view per well. Alexa 488 was used to label Bodipy 493 / 503, and Hoechst 33342 was used to label the cell nucleus for lipid accumulation fluorescence data collection.

[0040] (7) Subsequently, the phenotypic data of the collected images were extracted using Harmony 4.9 software, where the data processing method for Bodipy fluorescent labeling was as follows: Find nuclei→Find cytoplasm-Alexa 488→Calculate intensity properties (Method: Standard). That is, the cell nucleus was labeled with the Hoechst 33342 channel, and then the single cell was located based on the labeled cell nucleus. The neutral lipid droplets in the cytoplasm were identified by Find cytoplasm using the Alexa 488 channel, and their fluorescence intensity was calculated and output in text data format.

[0041] (8) GraphPad Prism 8.0 software was used to analyze the experimental results. One-way ANOVA was used to test the significant differences between the data, with p < 0.05 indicating statistical significance. The results were expressed as mean ± standard deviation (mean ± SD).

[0042] 4. Detection of lipid accumulation in HepG2 cells by Nile red staining

[0043] The cells were cultured in six-well plates. The modeling and drug administration procedures were the same as Bodipy staining. After 24 hours of drug administration and incubation, the original culture medium was carefully discarded and the cells were washed once with PBS. 200 μL of PBS buffer was added to each well, and then Nile red staining solution was added to make the final concentration in the culture medium 2.5 μg / mL. Oscillate and mix on a well plate oscillator, and let stand for 15 minutes in the dark. The results were observed and photographed under a Nikon inverted microscope with an excitation wavelength of 485nm-543nm. The IOD values ​​of the photos taken were integrated using ImageJ. GraphPad Prism 8.0 software was used to count and analyze the experimental results. The One-way ANOVA method was used to test and analyze the significant differences between the data, with p<0.05 indicating statistical significance, and the results were expressed as mean ± standard deviation (mean ± SD).

[0044] 3. Experimental Results

[0045] PA is the most abundant type of saturated fatty acid in plasma, and is associated with lipotoxicity in pancreatic β cells, hepatocytes, and many other cells, and is a key inducer of lipid accumulation in tissue cells. In lipid metabolism research, lipophilic dyes such as Nile red and BODIPY are often used to analyze the lipid content in tissue cells. The present invention constructs a hepatocyte lipid accumulation cell model of HepG2 cells induced by PA, and uses Bodipy and Nile red staining to analyze the cellular lipid content.

[0046] The results of HepG2 cell Bodipy staining are shown in Figure 1 As shown in Table 1. Compared with the control group, the model group had significantly larger and more green lipid droplet spots; compared with the model group, the lipid droplets in the BA and DBA groups were significantly reduced, and the improvement in the DBA group was particularly obvious. Figure 2 As shown in Table 1, compared with the control group, obvious orange-red neutral lipids appeared in the cells of the model group; compared with the model group, the neutral lipids in the cells of the BA and DBA groups were significantly reduced, and the improvement in the DBA group was particularly obvious.

[0047] These experimental results show that DBA can significantly improve PA-induced lipid accumulation in HepG2 cells; although BA can also improve PA-induced lipid accumulation in HepG2 cells, the effect is far less than that of DBA (under the same experimental conditions, the BA administration concentration needs to be increased to 40 μM to obtain an inhibitory effect close to that of 10 μM DBA).

[0048] Those skilled in the art know that the only difference between dihydrobaicalin and baicalin is a double bond, and the activities of the two should be similar. However, the effect of dihydrobaicalin in inhibiting lipid accumulation in HepG2 cells is much stronger than that of baicalin. This technical effect is unexpected to those skilled in the art.

[0049] Table 1 Cell staining results of each group

[0050] index Blank group (Group C) Model group (M group) Baicalin group (BA group) Dihydrobaicalin group (DBA group) HepG2-Bodipy 0.304±0.015 0.758±0.0794 0.515±0.089 0.240±0.036 HepG2-Nile Red 0.974±0.038 2.250±0.147 1.641±0.131 1.017±0.026

[0051] Example 2: Cell model based on mouse liver primary cells

[0052] 1. Experimental Materials

[0053] Dihydrobaicalin (DBA, Chengdu Pusi Biotechnology Co., Ltd., 56226-98-3);

[0054] C57 mice (Hangzhou Ziyuan Experimental Animal Technology Co., Ltd.);

[0055] Collagenase type IV (Sigma, C5138-1G);

[0056] HBSS buffer (Gibco, C14175500BT);

[0057] 10×PBS (Keygen, KGB50011);

[0058] Percoll cell separation medium (White Shark, BS909);

[0059] Australian fetal bovine serum (FBS, Gibco, 10270-106);

[0060] Pancreatin (Gibco, 25200072);

[0061] DMEM high-glucose medium (containing double antibodies) (Keygene, KGM12800-500);

[0062] Bodipy 493 / 503 fluorescent probe (Thermo, D3922);

[0063] Nile red dye (Thermo, N1142);

[0064] Fetal bovine serum (FBS, Gibco, 10270-106);

[0065] Pancreatin (Gibco, 25200072).

[0066] 2. Experimental Methods

[0067] 1. Cell acquisition and culture

[0068] This experiment uses the perfusion method to extract mouse liver parenchymal cells, with a purity of more than 90% and good cell viability. The specific steps are as follows:

[0069] (1) All surgical instruments were sterilized at high temperature before use. The mice were anesthetized with isoflurane, disinfected with 75% alcohol, and the limbs and head of the mice were fixed.

[0070] (2) After disinfecting the mouse abdomen, dissect the abdomen to expose the mouse liver and inferior vena cava. Insert a needle into the inferior vena cava and ligate it.

[0071] (3) All perfusion fluids are maintained at 37°C. First, fluid A is pumped in. A small incision is made in the portal vein and the fluid is perfused into the liver, which turns into a yellowish-brown earth color.

[0072] (4) The liver was digested and softened by perfusion with buffer B containing 0.5% collagenase IV, and then cut off the liver and placed in a culture medium containing 10% FBS.

[0073] (5) Tear the liver with forceps or cut it into pieces with scissors, filter it through a cell sieve (100 μm), and discard the upper filter residue.

[0074] (6) Centrifuge at 50 g for 3 min, discard the supernatant, resuspend in DMEM, and repeat once.

[0075] (7) Separate the cells using Percoll cell separation buffer at 1200 g for 10 min and discard the supernatant.

[0076] (8) Finally, mix the cells by pipetting with culture medium containing 10% FBS.

[0077] (9) Cell density is controlled at (5-10) 10 / mL.

[0078] (10) After inoculating the cells, spread them evenly and place them in a cell culture incubator for 4 h. After changing the medium, proceed to the next step of the experiment according to experimental needs.

[0079] 2. Detection of lipid accumulation in mouse liver primary cells by Bodipy staining

[0080] The grouping and operation methods are the same as “Bodipy staining to detect lipid accumulation in HepG2 cells”.

[0081] 3. Detection of lipid accumulation in mouse primary liver cells by Nile red staining

[0082] The grouping and operation methods are the same as “Nile red staining to detect lipid accumulation in HepG2 cells”.

[0083] 3. Experimental Results

[0084] In order to further determine the effect of DBA on lipid accumulation in hepatocytes, we extracted primary liver cells from mice for verification experiments. The results of Bodipy staining of primary liver cells from mice are shown in Figure 2. Figure 3 As shown in Table 2, compared with the control group, the model group had significantly larger and more green lipid droplet spots; compared with the model group, the lipid droplets in the BA and DBA groups were significantly reduced, and the improvement in the DBA group was particularly obvious. Figure 4 As shown in Table 2. Compared with the control group, obvious orange-red neutral lipids appeared in the cells of the model group; compared with the model group, the neutral lipids in the cells of the BA and DBA groups were significantly reduced, and the improvement in the DBA group was particularly obvious.

[0085] These experimental results show that DBA can significantly improve PA-induced lipid accumulation in primary liver cells; although BA can also improve PA-induced lipid accumulation in primary liver cells, the effect is far less than that of DBA (under the same experimental conditions, the BA administration concentration needs to be increased to 40 μM to obtain an inhibitory effect close to that of 10 μM DBA).

[0086] Those skilled in the art know that the only difference between dihydrobaicalin and baicalin is a double bond, and the activities of the two should be similar. However, the effect of dihydrobaicalin in inhibiting lipid accumulation in primary liver cells is much stronger than that of baicalin. This technical effect is unexpected to those skilled in the art.

[0087] Table 2 Cell staining results of each group

[0088] index Blank group (Group C) Model group (M group) Baicalin group (BA group) Dihydrobaicalin group (DBA group) Liver Primary-Bodipy 1.070±0.095 5.923±1.051 3.949±0.408 1.634±0.336 Liver Primary-Nile Red 6.488±0.607 10.440±1.110 8.883±0.473 6.455±0.437

[0089] Hepatocyte lipid accumulation can lead to the occurrence of a variety of diseases including non-alcoholic fatty liver disease and fatty liver disease. The present invention proves that DBA can effectively inhibit hepatocyte lipid accumulation, has the prospect of being developed into a lipid-lowering drug that inhibits hepatocyte lipid accumulation, and can be used to prevent and treat diseases caused by hepatocyte lipid accumulation such as non-alcoholic fatty liver disease and fatty liver disease. Those skilled in the art know that dihydrobaicalin and baicalin differ only by one double bond, and their activities should be similar, but the effect of dihydrobaicalin in inhibiting lipid accumulation in primary liver cells is much stronger than that of baicalin, and this technical effect is unexpected by those skilled in the art.

[0090] The purpose of the above-mentioned embodiments is to specifically introduce the essential content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to the specific embodiments.

Claims

1. Application of dihydrobaicalin in the preparation of drugs for treating fatty liver.

2. The use according to claim 1, characterized in that: The medicine uses dihydrobaicalin as an active ingredient and is prepared into a pharmaceutically acceptable dosage form using pharmaceutically acceptable excipients.

3. The use according to claim 2, characterized in that: The auxiliary material is solid, liquid or semi-solid auxiliary material.

4. The use according to claim 2, characterized in that: The dosage forms include tablets, capsules and soft gelatin capsules.

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