Application of lotus plumule extract in preparation of medicine for preventing and / or treating non-alcoholic fatty liver disease

Abstract

The invention discloses application of a lotus plumule extract in preparation of a medicine for preventing and / or treating a non-alcoholic fatty liver disease, and belongs to the technical field of separation and purification of Chinese herbal medicines. The lotus plumule extract prepared by the invention can relieve liver fatty degeneration and inflammatory response induced by HFD and relieve liver injury of NAFLD mice. The invention discusses the intervention effect and regulation mechanism of lotus plumule on NAFLD liver inflammation, provides more thoughts and directions for clinical prevention and treatment of NAFLD, and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of separation and purification of Chinese herbal medicines, and particularly relates to the use of lotus seed core extract in the preparation of drugs for preventing and / or treating non-alcoholic fatty liver disease. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) is a clinically syndrome induced by multiple factors, and its core pathological manifestation is excessive lipid deposition in hepatocytes. NAFLD includes three typical pathological subtypes: simple steatosis, non-alcoholic steatohepatitis (NASH) and fibrosis. Its influence is not limited to the liver itself, but also involves pathological changes in multiple organs such as the cardiovascular system, endocrine metabolism, etc. Clinical studies have shown that NAFLD may not only lead to end-stage liver diseases such as liver cirrhosis and liver cancer, but also has a significant association with type 2 diabetes, chronic kidney disease and specific extrahepatic malignancies. Epidemiological data shows that the global prevalence of NAFLD has reached 30.2%, and this growth trend is closely related to the prevalence of metabolic syndrome and lifestyle changes. Given that NAFLD has posed a major threat to public health, it is of great clinical significance to deeply explore its pathological mechanism and develop effective treatment strategies.

[0003] For such a complex metabolic disease as NAFLD, currently, regulatory drugs are mainly used in clinical treatment, and there is no specific treatment drug. Western medicine advocates the use of hypoglycemic, lipid-lowering, blood pressure-lowering, liver-protecting drugs such as insulin sensitizers, angiotensin 2 receptor antagonists, statin drugs, etc. However, most of these drugs are applied to cardiovascular diseases, and the treatment effect on the histological lesions of NAFLD liver is not obvious. Moreover, Western medicines have a single component and a clear target, and often have adverse reactions.

[0004] Therefore, developing effective and safe drugs for treating NAFLD liver injury has great research value for the clinical prevention and treatment of NAFLD. Summary of the Invention

[0005] In order to solve the above problems existing in the prior art, the purpose of the present invention is to provide the use of lotus seed core extract in the preparation of drugs for preventing and / or treating non-alcoholic fatty liver disease.

[0006] In order to achieve the above purpose, the present invention adopts the following technical scheme:

[0007] The present invention provides the use of lotus seed core extract in the preparation of drugs for preventing and / or treating non-alcoholic fatty liver disease.

[0008] Further, the pathological subtype of the non-alcoholic fatty liver disease is non-alcoholic steatohepatitis.

[0009] Further, the lotus seed core extract is obtained by the following method:

[0010] (1) Crush lotus seed plumule to obtain lotus seed plumule powder;

[0011] (2) Add a solvent to the lotus seed plumule powder obtained in step (1) to dissolve, extract, and collect the supernatant;

[0012] (3) Concentrate, defat, and dry the supernatant obtained in step (2) to obtain the lotus seed plumule extract.

[0013] Further, the lotus seed plumule powder described in step (1) is obtained by crushing lotus seed plumule and then sieving.

[0014] Further, the sieving is through a 60-mesh sieve.

[0015] Further, the solvent described in step (2) is an organic solvent, and the volume-mass ratio of the solvent to the lotus seed plumule powder is (1 - 10) L:1 kg.

[0016] Further, the organic solvent is ethanol, and the volume-mass ratio of the solvent to the lotus seed plumule powder is 5 L:1 kg.

[0017] Further, the ethanol is 80% ethanol.

[0018] Further, the extraction in step (2) is first ultrasonic extraction and then heating reflux extraction, repeated 1 - 5 times; the power of the ultrasonic extraction is 200 - 800 W, the time of the ultrasonic extraction is 10 - 60 min; the time of the reflux extraction is 0.5 - 2 h.

[0019] Further, the repetition in step (2) is repeated 2 times; the power of the ultrasonic extraction is 500 W, the time of the ultrasonic extraction is 30 min; the time of the reflux extraction is 1 h.

[0020] Further, the concentration in step (3) is vacuum concentration, and the defatting is defatting with petroleum ether.

[0021] Further, the drug is a drug for treating liver injury.

[0022] The present invention has achieved the following beneficial effects:

[0023] The present invention provides a lotus seed plumule extract, which as a whole can reduce HFD-induced hepatic steatosis and inflammatory response and alleviate liver injury in NAFLD mice. The present invention explores the intervention effect and regulatory mechanism of lotus seed plumule on NAFLD liver inflammation, provides more ideas and directions for clinical prevention and treatment of NAFLD, and has good application prospects.

[0024] The present invention further studied the extraction rates of lotus seed embryo extracts obtained under different extraction methods (reflux extraction, ultrasonic extraction), extraction solvents (water, ethanol with different volume concentrations (20%, 40%, 60%, 80%, 100%)), and solid-liquid ratios (1:5, 1:10, 1:20), and found that the extraction rate of the lotus seed embryo extract obtained by the method of Example 1 was the highest, and the overall peak shape and resolution were the best.

[0025] Obviously, based on the above content of the present invention, according to the common general knowledge and conventional means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions or changes can be made.

[0026] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. Brief Description of the Drawings

[0027] Figure 1 Effects of lotus seed embryo extract on the body weight of HFD-induced NAFLD mice. (A) Body weight of mice; (B) Epididymal fat weight of mice; (C) Epididymal fat index of mice. ** P<0.01 NC vs. HFD; # P<0.05 HFD+ENP vs. HFD.

[0028] Figure 2 Effects of lotus seed embryo extract on the liver morphology and liver index of NAFLD mice. (A) Liver morphology diagram; (B) Liver weight; (C) Liver index; (D) Small animal ultrasound view; (E) Kidney-liver ratio; (F) Percentage of liver fat content. **P<0.01 NC vs. HFD; #P<0.05 HFD+ENP vs. HFD; ##P<0.01 HFD+ENP vs. HFD.

[0029] Figure 3 Effects of lotus seed embryo extract on liver injury of NAFLD mice. (A) Liver pathological staining diagram; (B) Oil red O staining statistics; (C) Masson staining collagen deposition statistics. **P<0.01 NC vs. HFD; ##P<0.01 HFD+ENP vs. HFD.

[0030] Figure 4 Effects of lotus seed embryo extract on blood lipids of NAFLD mice. ** P<0.01 NC vs. HFD; # P<0.05 HFD+ENP vs. HFD; ##P < 0.01 HFD + ENP vs. HFD。

[0031] Figure 5 Effect of lotus plumule extract on liver function in NAFLD mice. * P < 0.05 NC vs. HFD; ** P < 0.01 NC vs. HFD; # P < 0.05 HFD + ENP vs. HFD; ## P < 0.01 HFD + ENP vs. HFD.

[0032] Figure 6 Effect of lotus plumule extract on various oxidative factors in liver tissues of NAFLD mice. (A) MDA content; (B) SOD content. ** P < 0.01 NC vs. HFD; ## P < 0.01 HFD + ENP vs. HFD. Figure 7 Effect of lotus plumule extract on various inflammatory cytokines in liver tissues of NAFLD mice. (A) TNF-α content; (B) IL-6 content; (C) IL-10 content. ** P < 0.01 NC vs. HFD; ## P < 0.01 HFD + ENP vs. HFD. Detailed implementation mode

[0033] The raw materials and equipment used in the present invention are all known products, obtained by purchasing commercially available products.

[0034] In the following experiments, if no description of temperature is made, it is a reaction under normal temperature conditions. Normal temperature means room temperature, which is 25 ± 5 °C.

[0035] In this study, SPSS 20.0 software was used for statistical analysis of data. All experimental data were measurement variables. For data groups conforming to normal distribution, parametric test methods were used: independent sample t-test for comparison between two groups, and one-way ANOVA for comparison among multiple groups. For data not conforming to normal distribution, Mann-Whitney U rank sum test in non-parametric test was used. The statistical significance threshold was set at P < 0.05, and all experimental procedures were verified by three independent repetitions to ensure the reliability of the results.

[0036] Example 1: Preparation of lotus plumule extract

[0037] The lotus plumule samples of the present invention were collected from Jianning County, Sanming City, Fujian Province in July 2023, and were taxonomically confirmed by Associate Professor Huang Zehao of Fujian University of Traditional Chinese Medicine as the embryonic appendages (dry young leaves and radicles) of the mature seeds of the plant Nelumbo nucifera Gaertn in the family Nymphaeaceae.

[0038] The lotus plumule was crushed and passed through a 60-mesh sieve, 5 times (L / kg) of 80% ethanol was added, and ultrasonic extraction was carried out for 30 min (500 W), followed by heating under reflux at 95 °C for 1 h. This was repeated twice, and the supernatant was collected, filtered. The filtrate was concentrated under reduced pressure using a rotary evaporator, and the concentrated solution was defatted with petroleum ether and dried under reduced pressure to obtain a powder, which was the lotus plumule extract.

[0039] In vivo drug preparation: According to the clinical dosage of lotus plumule for humans in the Chinese Pharmacopoeia, which is 5 g / d, and the drug extraction yield of 15.38%, and according to the dose conversion between humans and mice in the Pharmacological Experiment Methodology, the dosage of lotus plumule extract for mice was: 1.02 g / kg / d and 0.51 g / kg / d. When used for animal gavage, it was prepared with normal saline and stored at 4 °C for later use.

[0040] The beneficial effects of the present invention are demonstrated by the following experimental examples.

[0041] Experimental Example 1: Treatment of HFD-induced NAFLD with lotus plumule extract

[0042] (1) Experimental animals

[0043] Male C57BL / 6 mice (19 - 20 g, 6 weeks old) were purchased from Shanghai SCXK Experimental Animal Co., Ltd. (SCXK (Shanghai) 2022 - 0004) and were housed in the SPF-class animal room of the Animal Experiment Center. During the breeding period, the temperature in the animal room was maintained at 20.8 - 24.3 °C, with a daily temperature difference < 4 °C. The relative humidity was 47.2 - 67.2%, and the air change was 15 times / h; in addition, the mice were exposed to a 12-h light / dark cycle, the bedding was changed every 2 - 3 days, and the mice could drink water and eat freely. Ethical review number: 2023072. All experimental animal operations strictly followed the relevant international ethical guidelines for the use and care of experimental animals and the guidelines of the National Institutes of Health of the United States.

[0044] (2) Animal grouping

[0045] Twenty-four 6-week-old male C57BL / 6 mice were randomly divided into four groups: control group (NC group), model group (HFD group), lotus seed core extract administration dose of 0.51 g / kg / d group (HFD+L-ENP), and lotus seed core extract administration dose of 1.02 g / kg / d group (HFD+H-ENP), with 6 mice in each group. The experimental animals were fed adaptively for 1 week before the formal experiment, and then were fed in groups: during the 20-week intervention period, the control group was given normal feed throughout the process, and the HFD group (HFD, high-fat diet) was fed with a high-fat modeling feed. Before feeding with the high-fat feed, the mice were fasted for 8-12 h to make them in a semi-starved state, and the water intake should be kept normal during this period. After 6 weeks of high-fat feeding, the success degree of the NAFLD model was evaluated. The lotus seed core extract administration groups were intragastrically administered with the corresponding administration dose once a day, and the remaining groups were given the same volume of normal saline for 14 consecutive weeks. The body weight was measured regularly every week to track the changes in physiological parameters.

[0046] I. Effects of lotus seed core extract on body weight and fat of HFD-induced NAFLD mice

[0047] The present invention evaluates the intervention effect based on the dynamic change data of the body weight of NAFLD model mice, as Figure 1 shown. At the 6th week of modeling, the body weight of the mice in the HFD group was significantly higher than that in the NC group, and there was a significant statistical difference between the two groups (P<0.01). Continuing the intervention until the 20th week, the lotus seed core extract administration groups (HFD+L-ENP group and HFD+H-ENP group) showed significant body weight regulation effects compared with the HFD group (P<0.05). It is worth noting that the epididymal fat weight of the mice in the lotus seed core extract administration groups was significantly reduced (P<0.01), and its fat index also showed a gradient downward trend with the increase of the dose, showing a clear dose-effect relationship.

[0048] II. Effects of lotus seed core extract on liver morphology of HFD-induced NAFLD mice

[0049] A Vevo (Vevo2100, visualsonic) small animal ultrasonic detector was used to detect the liver-kidney ratio and liver fat content of the mice in each group. 24 h before the detection, the mice were fasted but not water-deprived. After the abdomen of the mice was depilated cleanly and fully exposed, they were anesthetized by inhalation of isoflurane. The coupling agent was applied to the chest and abdomen of the mice, and then non-invasive liver function determination of the mice was performed using a small animal ultrasound, and the probe frequency was 30 MHz. In this study, the liver ultrasound images were labeled with reference to the results calculated by the existing manual method of the liver fat content (Liver fat content, LFC) of the control, and were used for the calculation of two indexes, namely, the liver-kidney ratio of ultrasound intensity and the liver attenuation rate of ultrasound intensity, after digital quantification of the images.

[0050] The references for the above-mentioned "existing manual method" are as follows: 1. YunSheng W, Xiao Y, GuiPing Z, et al. Assessment of hepatic fat content in using quantitative ultrasound measurement of hepatic / renal ratio and hepatic echo - intensity attenuation rate. [J]. Medical ultrasonography, 2020, 22(4): 393 - 401. 2. Ming - Feng X, Hong - Mei Y, Wan - Yuan H, et al. Standardized ultrasound hepatic / renal ratio and hepatic attenuation rate to quantify liver fat content: an improvement method. [J]. Obesity (Silver Spring, Md.), 2012, 20(2): 444 - 52. For the annotation of the hepatic - renal ratio image, the ROIs (Regions of Interest) of the liver and kidney need to be annotated separately. Select and calculate the gray - scale values of two ROIs with the same size of 2.25 cm at the proximal and distal ends of the anterior margin of the liver (An and Af; An is proximal, Af is distal) for calculating the attenuation rate. The calculation formula for the attenuation rate a is: a = (lnAn - lnAf) / [2 × (△d × f)]. Substitute into the calculation formula for liver fat content to obtain the liver fat content. The calculation formula is as follows: Liver fat content (percentage) = 62.592 × ultrasonic hepatic - renal ratio + 168.076 × ultrasonic attenuation rate - 27.863. Use Image J software to analyze the hepatic - renal ratio and liver fat content of mice. Each mouse is measured 3 times and then statistical analysis is performed. 2 The liver morphology and related indicators of each group of mice were detected, and the data showed (

[0054] The liver morphology and related indicators of each group of mice were detected, and the data showed ( Figure 2) Compared with the NC group, the livers of mice in the HFD group showed uniform swelling and a light yellow appearance, and their liver indices increased significantly (P<0.01); while the liver morphology and indices of the HFD+ENP group (HFD+L-ENP group and HFD+H-ENP group) showed a trend of approaching the NC group level, and the results were statistically significant. The small animal ultrasound results indicated that ENP treatment could reduce the kidney-liver ratio and liver fat content in NAFLD mice ( Figure 2 D-F).

[0055] III. Effects of lotus seed core extract on liver injury in HFD-induced NAFLD mice

[0056] 1. Observation of lipid droplet deposition in the livers of NAFLD mice by Oil Red O staining

[0057] After 14 weeks of drug administration to the mice, animal samples were taken. First, blood was collected by orbital puncture, centrifuged, and the supernatant was collected for later use. After the mice were sacrificed, epididymal fat tissue and liver tissue were isolated from the mouse samples and weighed respectively. The excised liver was washed with pre-cooled PBS and cut into two parts. One part was frozen in liquid nitrogen and transferred for Oil Red O staining and Western blot analysis, and the other part of the sample was fixed in 4% paraformaldehyde solution for subsequent H&E and Masson staining experiments.

[0058] (1) OTC embedding

[0059] The mouse liver tissue samples were quickly transferred into a pre-cooled freezing mold, and then an appropriate amount of OCT embedding medium (cryostat embedding medium) was evenly dropped to ensure complete coverage of the tissue. The mold was placed in a pre-cooled dry ice-isopropanol mixture for rapid freezing. After complete freezing, the embedded tissue was obtained and stored at -80°C for freezing preservation.

[0060] (2) Tissue sectioning

[0061] The embedded tissue obtained in step (1) was taken out, and liver sections with a thickness of 8-10 μm were cut using a cryostat to obtain tissue sections. The sections were placed on glass slides and used after drying and fixation.

[0062] (3) Staining

[0063] The tissue sections prepared in step (2) were fixed with 4% paraformaldehyde for 10 min, then washed three times with distilled water; then soaked in 60% isopropanol for 20-30 s; stained with Oil Red O staining working solution for 15 min; after staining, washed three times again with distilled water; counterstained with Mayer hematoxylin staining solution for 2 min for the cell nuclei; finally, the samples were thoroughly rinsed with distilled water; after absorbing the residual moisture, the slides were sealed with glycerin jelly and observed and analyzed under a microscope.

[0064] 2. Observe the pathological changes of the liver in NAFLD mice by H&E staining

[0065] (1) Dehydration and embedding

[0066] Gradually dehydrate the liver fixed as described above in the "Another part of the sample was fixed in 4% paraformaldehyde solution": 70% absolute ethanol for 40 min; 80% absolute ethanol for 40 min; 90% absolute ethanol for 40 min; 95% absolute ethanol for 40 min; 100% absolute ethanol, first channel for 10 min; 100% absolute ethanol, second channel for 10 min; xylene, first channel for 15 min; xylene, second channel for 10 min; paraffin, first channel for 10 min; paraffin, second channel for 10 min; paraffin, third channel for 25 min, and then perform tissue embedding.

[0067] (2) Section dehydration

[0068] Trim the paraffin sample of liver tissue into 4-μm-thick sections with a microtome, transfer it to a 37°C constant-temperature water bath for wrinkle stretching treatment. After the sample is completely flattened, transfer it to a 60°C constant-temperature slide dryer for continuous heat curing for 2 - 3 hours, and then perform gradient dewaxing: xylene, first channel for 15 min; xylene, second channel for 15 min; 100% absolute ethanol, first channel for 5 min; 100% absolute ethanol, second channel for 5 min; 95% absolute ethanol for 5 min; 90% absolute ethanol for 5 min; 80% absolute ethanol for 5 min; 70% absolute ethanol for 5 min. Then wash it 3 times in ultrapure water, 5 min each time, and ensure that the water is clear each time during the water washing process.

[0069] (3) Staining

[0070] Immerse the washed liver tissue sections in hematoxylin stain for 3 min to stain the cell nuclei. Then use pure water to perform a 10-min blueing treatment on the slides, and transfer them to eosin stain to complete 1-min cytoplasmic staining. When mounting the slides, evenly drop neutral balsam on the outer edge of the tissue sample, cover the cover slip, and place it in a ventilated environment to dry naturally. Finally, observe and analyze the liver tissue structure by microscopic imaging system for pathological observation and image acquisition.

[0071] 3. Observe the collagen fiber deposition in the liver tissue of NAFLD mice by Masson staining

[0072] The liver tissue sections were stained with hematoxylin for nuclear staining for 10 min, then differentiated with 1% hydrochloric acid alcohol, washed with pure water, and placed in Ponceau S staining solution for 8 min, treated in 2% acetic acid aqueous solution for 2 min, then differentiated in 1% phosphoric acid aqueous solution for 4 min and stained with aniline blue for 2 min, and then washed with 2% acetic acid aqueous solution for 1 min and washed with pure water three times. The samples were successively dehydrated in 95% ethanol I (5 min) and absolute ethanol I (5 min), then made transparent in xylene I (5 min) and xylene II (5 min), and then sealed with neutral gum. Subsequently, the sections were observed and photographed under an optical microscope.

[0073] Oil red O, HE and Masson staining were used to observe the pathological changes of liver cells, and the results were as Figure 3 shown. The hepatic lobule structure of the NC group remained intact, and the hepatocytes were arranged orderly; compared with the NC group, the HFD group showed obvious pathological changes. Oil red O staining showed diffuse lipid droplet deposition, and HE staining showed extensive infiltration of inflammatory cells, fatty degeneration of hepatocytes accompanied by cytoplasmic vacuoles and ballooning degeneration, suggesting severe fatty degeneration and inflammatory reaction. In contrast, the pathological damage of the liver tissue in the lotus seed core extract administration groups (HFD+L-ENP group and HFD+H-ENP group) was significantly alleviated. In addition, Masson staining showed that there was no significant difference in collagen deposition among the groups, suggesting that the process of liver fibrosis had not been initiated.

[0074] This experimental example proved that the lotus seed core extract of the present invention has a significant therapeutic effect on liver injury in HFD-induced NAFLD mice.

[0075] IV. Effects of lotus seed core extract on blood lipids in HFD-induced NAFLD mice

[0076] After the whole blood samples of mice were left standing at room temperature for 2 hours, they were centrifuged at 3000 rpm for 15 min at 2-8 °C, and the supernatant was collected, aliquoted and stored at -80 °C. During this period, attention should be paid to avoiding repeated freezing and thawing of the samples. In the detection preparation stage, the thawed samples should be centrifuged again. An automatic biochemical analyzer was used to detect the serum samples, and the lipid metabolism indexes included triglyceride (TG), total cholesterol (TC), high and low density lipoprotein cholesterol (HDL-C, LDL-C), and the operation was carried out according to the kit instructions.

[0077] The regulatory effect of lotus seed core extract on lipid metabolism in NAFLD mice was evaluated by detecting the levels of serum TG, TC, HDL-C and LDL-C, and the results were as Figure 4As shown, the levels of serum TG, TC, and LDL-C in the HFD group were significantly higher than those in the NC group, while the level of HDL-C decreased, with obvious statistical significance (P<0.01), indicating that a high-fat diet successfully induced dyslipidemia; compared with the HFD group, the contents of TG, TC, and LDL-C in the lotus seed core extract administration group decreased significantly, and the level of HDL-C increased significantly (P<0.05).

[0078] V. Effects of lotus seed core extract on liver function of HFD-induced NAFLD mice

[0079] After leaving the whole blood sample of the little nurse to stand at room temperature for 2 hours, centrifuge it at 3000 revolutions per minute for 15 minutes at 2-8°C, collect the supernatant, divide it into aliquots and store it at -80°C. During this period, care should be taken to avoid repeated freezing and thawing of the sample. In the detection preparation stage, the thawed sample should be centrifuged again. Use an automatic biochemical analyzer to detect the serum sample. The liver function indicators include alanine aminotransferase (ALT), aspartate aminotransferase (AST), direct bilirubin (DBIL), total bilirubin (TBIL), total bile acid (TBA), and alkaline phosphatase (ALP), and operate according to the kit instructions.

[0080] Evaluate the improvement effect of lotus seed core extract on liver injury in NAFLD mice by detecting the levels of serum ALT, AST, DBIL, TBIL, TBA, and ALP in mice. The results are as Figure 5 shown. The levels of all indicators in the HFD group were significantly increased, indicating liver function damage in NAFLD mice; after administration of lotus seed core extract, the levels of all indicators decreased significantly, and the difference was statistically significant (P<0.05).

[0081] VI. Effects of lotus seed core extract on various oxidative factors in the liver tissues of NAFLD mice

[0082] Liver tissue sample processing procedure: Take 10 mg of liver samples from each group and place them in 1.5 mL centrifuge tubes, and add 2 stainless steel magnetic beads to each tube. Fix the sample tubes in a grinding module pre-cooled to 4°C, and use a tissue homogenizer for mechanical fragmentation. Set the parameters as a vibration frequency of 60 Hz and a continuous action of 90 seconds. After lysis, centrifuge at 3000 r / min for 3 minutes, and collect the supernatant in the centrifuge tube as the working mother liquor, which is diluted 10 times during experimental detection.

[0083] Detect the content of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) in liver tissues by ELISA method to evaluate the regulatory effect of lotus seed core extract on oxidative stress. The results are as Figure 6Shown as follows: The group administrated with lotus plumule extract could significantly inhibit the abnormally increased MDA in the liver tissue induced by HFD (P<0.01), and at the same time effectively up-regulate the SOD activity. Its regulatory effect was significantly negatively correlated with the degree of liver lipid deposition. The above results indicated that this extract could effectively alleviate the liver oxidative stress state and then relieve the abnormal lipid accumulation by enhancing the endogenous antioxidant defense system.

[0084] VII. Effects of lotus plumule extract on various inflammatory cytokines in the liver tissue of NAFLD mice

[0085] Lipid overload induced by HFD directly triggers oxidative stress, and oxidative damage activates the inflammatory cascade reaction, triggering the release of pro-inflammatory factors. The cytokine imbalance exacerbates lipid metabolism disorders, forming a vicious cycle of the pathological progression of NAFLD.

[0086] Liver tissue sample processing procedure: Take 10 mg of liver samples from each group and place them in 1.5 mL centrifuge tubes, and add 2 stainless steel magnetic beads to each tube. Fix the sample tubes in the grinding module pre-cooled to 4°C, and perform mechanical crushing treatment with a tissue homogenizer. Set the parameters as the vibration frequency of 60 Hz and continuously act for 90 seconds. After completion of lysis, centrifuge at 3000 r / min for 3 minutes, collect the supernatant in the centrifuge tube as the working mother liquor, and dilute it 10 times during experimental detection.

[0087] The levels of inflammatory factors in the mouse liver were detected by ELISA method, and the results were as Figure 7 shown as follows: The group administrated with lotus plumule extract significantly reversed the abnormally increased TNF-α and IL-6 induced by HFD (P<0.01), and at the same time improved the down-regulation of IL-10 level. This effect was significantly negatively correlated with the degree of liver pathological damage, confirming that lotus plumule extract could effectively relieve the liver inflammatory state by regulating the inflammatory factor network.

[0088] The above results together indicated that the NAFLD induced by HFD in this experiment had reached the subtype stage of non-alcoholic steatohepatitis (NASH). Administration of the lotus plumule extract of the present invention could alleviate the liver steatosis and inflammatory reaction induced by HFD and reduce the liver injury in NAFLD mice.

[0089] In summary, the lotus plumule extract prepared by the present invention could alleviate the liver steatosis and inflammatory reaction induced by HFD and reduce the liver injury in NAFLD mice. The present invention explored the intervention effect and regulatory mechanism of lotus plumule on NAFLD liver inflammation, providing more ideas and directions for the clinical prevention and treatment of NAFLD, and having good application prospects.

Claims

1. Use of lotus plumule extract in the preparation of a medicament for preventing and / or treating non-alcoholic fatty liver disease.

2. The use according to claim 1, wherein: The lotus plumule extract is obtained by the following method: (1) Crush lotus plumule to obtain lotus plumule powder; (2) Add a solvent to the lotus plumule powder obtained in step (1) to dissolve, extract, and collect the supernatant; (3) Concentrate, defat, and dry the supernatant obtained in step (2) to obtain the lotus plumule extract.

3. The use according to claim 2, wherein: The lotus plumule powder in step (1) is obtained by crushing lotus plumule and then sieving; preferably, the sieving is through a 60-mesh sieve.

4. According to the use described in claim 2, wherein: The solvent in step (2) is an organic solvent, and the volume-mass ratio of the solvent to the lotus plumule powder is (1-10) L:1 kg; preferably, the organic solvent is ethanol, and the volume-mass ratio of the solvent to the lotus plumule powder is 5 L:1 kg; more preferably, the ethanol is 80% ethanol.

5. The use according to claim 2, characterized in that: The extraction in step (2) is first ultrasonic extraction and then heat reflux extraction, repeated 1-5 times; the power of the ultrasonic extraction is 200-800 W, the time of the ultrasonic extraction is 10-60 min; the time of the reflux extraction is 0.5-2 h.

6. According to the use described in claim 5, it is characterized in that: The repetition in step (2) is repeated 2 times; the power of the ultrasonic extraction is 500 W, the time of the ultrasonic extraction is 30 min; the time of the reflux extraction is 1 h.

7. The use according to claim 2, wherein: The concentration in step (3) is vacuum concentration, and the defatting is defatting with petroleum ether.

8. The use according to any one of claims 1 to 7, characterized in that: The medicament is a medicament for treating liver injury.

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