Application of allium mongolicum extract

Abstract

The invention provides application of an allium mongolicum extract. The allium mongolicum extract is used for preparing a feed for improving metabolism-related fatty liver diseases. The allium mongolicum extract disclosed by the invention is derived from natural plant compounds, has relatively high safety, can be used for reducing blood sugar and insulin levels of serum, reducing biochemical index levels of the serum and improving fatty degeneration of the liver, and exerts a positive protection effect on metabolism-related fatty liver diseases by cooperatively regulating lipid metabolism-related genes; the method has guiding significance for subsequent further development and utilization.

Description

Technical Field

[0001] This invention belongs to the field of lipid-lowering feed technology, specifically relating to the application of a sand onion extract. Background Technology

[0002] Metabolic dysfunction-associated fatty liver disease (MAFLD) is one of the most prevalent chronic liver diseases worldwide in recent years. MAFLD is a relatively new disease concept, formerly known as non-alcoholic fatty liver disease (NAFLD). Its core characteristic is excessive fat accumulation in the liver, accompanied by insulin resistance (IR) or metabolic dysfunction. Against the backdrop of rising prevalence of obesity, type 2 diabetes mellitus (T2DM), and metabolic syndrome (MetS), the incidence of MAFLD is also showing a year-on-year upward trend. Statistics show that the global prevalence of MAFLD is approximately 33%; in my country, the prevalence is approximately 29.6%, with the prevalence reaching as high as 66.2% in obese patients and 51.8% in patients with T2DM. Notably, the age of onset of MAFLD is showing a trend towards younger ages, with the global prevalence of MAFLD in children increasing by 0.26% annually. If effective interventions for metabolic dysfunction are not implemented in a timely manner, the disease burden of MAFLD in my country will continue to rise rapidly in the future.

[0003] High-fructose, high-fat diets play a crucial role in the development and progression of MAFLD. In recent years, fructose consumption has increased rapidly. As a monosaccharide, it is a major component of the most widely used sweeteners and is abundant in various industrially manufactured foods. Excessive fructose intake is considered one of the causes of visceral obesity and insulin resistance (IR), potentially impairing liver function and leading to MetS and MAFLD. Saturated fatty acids and trans fatty acids in a high-fat diet further exacerbate hepatic steatosis and inflammation, causing liver damage. Animal experiments have shown that long-term high-fructose, high-fat diets can induce typical pathological features of MAFLD in rats, such as hepatic steatosis, inflammation, and increased insulin resistance. Early development is a critical period for metabolic programming, during which the body is more sensitive to environmental factors. Studies by Oliveira et al. have shown that a high-fat diet for mother mice increases the susceptibility of offspring to MAFLD in adulthood, while fructose intake during puberty in offspring exacerbates pre-existing liver metabolic damage. MAFLD occurring in childhood may significantly increase the risk of cardiovascular disease, type 2 diabetes mellitus (T2DM), chronic kidney disease, and liver cancer in adulthood.

[0004] Currently, the clinical management of MAFLD mainly relies on lifestyle interventions (such as diet control and exercise) and medications targeting metabolic comorbidities. However, patient adherence to lifestyle interventions is generally poor, and long-term medication may lead to risks such as growth inhibition and gastrointestinal adverse reactions. Finding safe and effective prevention and treatment methods has become a current research hotspot. Natural compounds, due to their wide availability, multiple targets, multiple mechanisms, and low toxicity, have shown great potential in the prevention and treatment of MAFLD. However, there are few reports on the effects of wild onion on the prevention and treatment of MAFLD, and whether it has a protective effect against MAFLD induced by high fructose and high-fat exposure in early development remains to be explored. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an application of scallion extract to address the shortcomings of the prior art. This scallion extract can be used to prepare feed to improve metabolic-related fatty liver disease, effectively reduce serum blood glucose, serum insulin and serum biochemical index levels, improve liver steatosis, and exert a protective effect on MAFLD rats by synergistically regulating lipid metabolism-related genes. This has guiding significance for further development and utilization.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: an application of sand onion extract, wherein the sand onion extract is used to prepare feed for improving metabolic-related fatty liver disease.

[0007] Preferably, the application includes one or more of the following applications: The sand onion extract is used to lower serum blood glucose levels; The sand onion extract is used to lower serum insulin levels; The sand onion extract is used to reduce serum biochemical levels. The scallion extract is used to improve hepatic steatosis; The scallion extract is used to reduce the mRNA expression level of genes related to liver fat metabolism.

[0008] Preferably, when the sand onion extract is used to lower serum blood glucose levels and serum insulin levels, it can significantly reduce the serum insulin resistance index.

[0009] Preferably, when the sand onion extract is used to reduce serum biochemical levels, the serum biochemical levels include TC, TG, and One or more.

[0010] Preferably, when the sand onion extract is used to reduce the mRNA expression level of liver lipid metabolism-related genes, the liver lipid metabolism-related genes include... , , and One or more of them.

[0011] Compared with the prior art, the present invention has the following advantages: The sand onion extract of this invention is derived from natural plant compounds and has a high safety profile. It can effectively improve insulin resistance and lipid metabolism disorders in MAFLD rats induced by high fructose, high lipid, and combined high fructose and high lipid in early life. It also exerts a protective effect on MAFLD rats by synergistically regulating lipid metabolism-related genes. This has guiding significance for further development and utilization, and provides an innovative perspective for early prevention and control of MAFLD in high-risk populations. It also helps to promote the development, promotion and utilization of the nutritional value of sand onion, a specialty of Northwest China.

[0012] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0013] Figure 1 This is a graph showing the changes in body weight of rats in each group in Example 1 of the present invention.

[0014] Figure 2 This is a comparison chart of serum blood glucose, insulin, and HOMA-IR index of rats in each group during weeks 7 and 12 of Example 1 of the present invention.

[0015] Figure 3 This is a comparison chart of serum biochemical indicators of rats in each group during week 7 in Example 1 of the present invention.

[0016] Figure 4 This is a comparison chart of serum biochemical indicators of rats in each group during week 12 in Example 1 of the present invention.

[0017] Figure 5 These are morphological images of the livers of rats in each group during week 12 of Example 1 of the present invention.

[0018] Figure 6 These are HE staining images of rat liver tissue from each group at week 7 and week 12 in Example 1 of the present invention.

[0019] Figure 7 The livers of rats in each group during week 7 of Example 1 of this invention. FAS , HSL , LPL , , , , , The level of expression.

[0020] Figure 8 The livers of rats in each group during week 12 of Example 1 of this invention. FAS, HSL , LPL, , , , , The level of expression.

[0021] Figure 9 The livers of rats in each group during week 7 of Example 1 of this invention. FAS , HSL , LPL, , , , , Protein expression level graph.

[0022] Figure 10 The livers of rats in each group during week 12 of Example 1 of this invention. FAS , HSL , LPL , , , , , Protein expression level graph. Detailed Implementation

[0023] Example 1 This example demonstrates the application of scallion extract in the preparation of feed to improve metabolic-related fatty liver disease.

[0024] I. Experimental Materials and Methods: (a) Experimental materials: Forty-eight clean-grade male Sprague-Dawley (SD) rats, 3 weeks old and weighing 60g-80g, were purchased from the Experimental Animal Center of Ningxia Medical University (Animal Production License No.: SYXK(Ning)2020-0001). The animal experiments were approved by the Medical Ethics Review Committee of Ningxia Medical University (2023-264), and the experimental process strictly followed the requirements of medical laboratory animal welfare ethics.

[0025] The sand onion extract used in the experiment was provided by Ningxia Xiangcao Biotechnology Co., Ltd.; the rat feed was produced and provided by Shanghai Shuyu Biotechnology Co., Ltd.

[0026] (II) Experimental Methods: 1. Grouping and administration of experimental animals: Rats were housed in a clean-grade animal room, with an environmental condition of 50% relative humidity and 23°C, and a 12-hour day-night cycle was maintained. During the experiment, rats in all groups had free access to food and water. During the acclimatization period, all rats were fed a maintenance diet (protein, fat, and carbohydrates provided 21%, 11%, and 68% of energy, respectively). After one week of acclimatization, rats were randomly divided into the following eight groups according to their body weight: ① Normal control group (NC, N=6): given distilled water and maintenance diet; ② Normal control-shallot group (NC-AMR, N=6): given distilled water and maintenance diet; ③ High fructose group (HF, N=6): given 10% fructose solution and maintenance diet; ④ High fructose-shallot group (HF-AMR, N=6): given 10% fructose solution and maintenance diet; ⑤ High fat group (HL, N=6): given distilled water and high fat diet (protein, fat, and carbohydrates provided 19.79%, 60.93%, and 19.27% ​​of energy, respectively); ⑥ High fat-shallot group (HL-AMR, N=6): given distilled water and high fat diet; ⑦ High fructose combined with high fat group (HFHL, N=6): given distilled water and high fat diet; ⑧ High-fructose combined with high-fat - scallion group (HFHL-AMR, N=6): Administered 10% fructose aqueous solution and high-fat diet. Eight groups of rats were given the corresponding diet and water. Two weeks after modeling, each scallion intervention group had 0.15 mL / g of scallion extract added to their original diet to prepare an intervention diet containing the equivalent of 1.5g of fresh scallion per gram of feed. Three rats from each group were randomly sacrificed at 7 and 12 weeks. The health status of the rats was observed and recorded during the experiment. The rats' food and water intake were monitored every two days, and their body weight was measured at fixed times each week.

[0027] 2. Sample collection and processing: At each time point in the experiment, SD rats were fasted for 12 hours. The following morning, after fasting and weighing, they were anesthetized in a closed induction chamber with 5% isoflurane. Blood was collected from the abdominal aorta after opening the abdominal cavity, centrifuged at 1356×g, and the serum was separated and stored at -80℃ for analysis. After euthanasia by cervical dislocation, tissue samples from the heart, liver, kidneys, and reproductive organs were quickly collected. The wet weight of each organ was accurately weighed using an electronic balance. After weighing, the samples were immediately aliquoted and stored at -80℃ for analysis. After weighing the liver, a 1.0cm×1.0cm tissue block from the same location was cut, placed in 4% paraformaldehyde fixative, and stored at 4℃ for subsequent histological analysis.

[0028] 3. Calculation of indicators: Energy intake (kcal / day) in the control group = energy from basal feed; energy intake (kcal / day) in the high-fructose group = energy from basal feed + fructose energy; energy intake (kcal / day) in the high-fat group = energy from high-fat feed; energy intake (kcal / day) in the high-fructose and high-fat group = energy from high-fat feed + fructose energy (Note: basal feed 3.639kcal / g, high-fat feed 4.926kcal / g, fructose energy 4.0kcal / g; organ coefficient (%) = organ weight (g) / body weight (g) × 100%).

[0029] 4. Determination of serum biochemical indicators: Serum total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C) ), LDL cholesterol ( Blood glucose concentration was measured using an enzyme-linked immunosorbent assay (ELISA) kit. Serum insulin levels were quantitatively analyzed using an ELISA kit. The homeostasis model assessment of insulin resistance (HOMA-IR) score was used to evaluate the degree of insulin resistance, calculated as follows: HOMA-IR = insulin (mIU / L) × fasting blood glucose (mmol / L) / 22.5.

[0030] 5. Histopathological observation of liver tissue: Preparation of tissue sections: ① Fixation: After anesthesia, rat liver tissue was dissected and a 5mm section was quickly cut. 3 ① Tissue blocks were fixed in pre-cooled 4% paraformaldehyde for 24 hours; ② Tissue collection: After removing the tissue from the fixative, it was trimmed into blocks using an ophthalmic scalpel under ventilated conditions to ensure regular shape. The trimmed tissue blocks and corresponding labels were then placed into embedding cassettes; ③ Dehydration and paraffin infiltration: Gradient alcohol dehydration: 75% alcohol for 4 hours, 85% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol for 1 hour, and anhydrous ethanol. Anhydrous ethanol , alcohols and benzene (8 min), xylene xylene 1. Melt paraffin I at 65℃ for 1 hour, melt paraffin II at 65℃ for 1 hour, melt paraffin III at 65℃ for 1 hour; 2. Paraffin embedding: The paraffin-impregnated tissue sample is placed in the embedding equipment for embedding. First, liquid paraffin is injected into the embedding mold. Before the paraffin is completely solidified, the tissue is taken out from the dehydration box and placed in the mold according to the sectioning direction. At the same time, a label with sample information is attached. Then the mold is moved to a low temperature environment of -20℃ for rapid solidification. After the paraffin is completely solidified, it is demolded and the edges of the wax block are trimmed; 3. Paraffin sectioning: The trimmed wax block is fixed in the paraffin microtome for sectioning. Thick sections were prepared, and then the sections were flattened in a 40°C constant temperature water bath. The tissue was then retrieved using a glass slide and transferred to a 60°C oven for baking. After the moisture had completely evaporated and the paraffin had fully melted, the slides were removed and cooled to room temperature for later use.

[0031] Staining steps: ① Dewaxing paraffin sections to water: Place the sections into xylene in sequence. xylene Anhydrous ethanol Anhydrous ethanol ① Slides were prepared by 1) 75% alcohol for 5 min, washed with tap water, and dried; ② Hematoxylin staining for 3 min, followed by water washing and differentiation with hydrochloric acid-alcohol solution for 2 s, then water washing and drying; ③ Hematoxylin-eosin staining for 6 s, followed by water washing for 3 s and dehydration with 100% alcohol for 2 s; ④ Mounting with neutral resin after drying; ⑤ Scanning and analyzing the slides using a high-resolution panoramic imaging system.

[0032] 6. Total RNA extraction and Real-time PCR detection: Total RNA extraction: Approximately 50 mg of rat liver tissue was weighed from each group, and cells were disrupted using liquid nitrogen cryogenic grinding. The ground product was transferred to a 1.5 mL RNase-free centrifuge tube, followed by the addition of 1 mL TRIzon reagent. Add [unclear text - likely a continuation of the previous sentence] to the centrifuge tube. After adding the chloroform solution, gently invert the centrifuge tube horizontally for 15 seconds to ensure thorough mixing. Then, incubate at room temperature for 5 minutes. Centrifuge at 12000 rpm for 15 minutes at 4°C. Clear layering is visible. Carefully pipette the upper colorless aqueous phase (approximately...) Avoid the intermediate protein layer, then transfer to a new RNase-free centrifuge tube; add an equal volume of isopropanol to the centrifuge tube, mix horizontally on a shaker, and then let stand at room temperature for 10 minutes; place the centrifuge tube in a refrigerated centrifuge and centrifuge at 4°C and 12,000 rpm for 10 minutes. After centrifugation, carefully remove the supernatant; a white RNA precipitate will be visible at the bottom of the tube. Carefully remove the supernatant from the centrifuge tube, add 1 mL of 75% ethanol solution to the centrifuge tube containing the RNA precipitate, and gently invert the centrifuge tube horizontally to mix the liquid thoroughly; centrifuge at 4°C and 12,000 rpm for 3 minutes; after thoroughly removing any remaining liquid from the centrifuge tube using a pipette, open the centrifuge tube and place it in a clean workbench to air dry for 6 minutes; add... Use RNase-free water and gently pipette to mix until the RNA is completely dissolved. After labeling the sample information on the centrifuge tube with a waterproof marker, immediately transfer it to a -70°C ultra-low temperature freezer for long-term storage to prevent RNA degradation.

[0033] RNA quality control: Nanodrop spectrophotometer was used to assess RNA quality: after instrument initialization and blank calibration (RNase-free water), samples were transferred... Close the detection arm at the detection platform (where droplets are evenly distributed) to measure the absorbance at 230 / 260 / 280 nm and record the data.

[0034] Reverse transcription: RNA was reverse transcribed into cDNA according to the reverse transcription kit (MonScript RTIII All-in-One Mix (with dsDNase), Mona Biotechnology Co., Ltd.)

[0035] Real-time PCR detection: The reaction system consisted of: MonAmp SYBR Green qPCR Mix 10µL, upstream primer ( ) Downstream primers ( ) 1 µL of cDNA template and ddH2O were added to a final volume of 20 µL. The reaction program was: 95 °C for 1 min; 95 °C for 15 s, 60 °C for 30 s, for 40 cycles. To ensure the accuracy and reliability of the experiment, all operations were performed on ice, using GAPDH as an internal control. The relative expression level of the target gene is calculated.

[0036] The nucleotide sequence of the upstream primer for the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene is shown in SEQ ID No. 1, and the nucleotide sequence of the downstream primer is shown in SEQ ID No. 2; the nucleotide sequence of the upstream primer for the FAS (lipase) gene is shown in SEQ ID No. 3, and the nucleotide sequence of the downstream primer is shown in SEQ ID No. 4. The nucleotide sequence of the upstream primer of the (hormone-sensitive lipase-1) gene is shown in SEQ ID No. 5, and the nucleotide sequence of the downstream primer is shown in SEQ ID No. 6; The nucleotide sequence of the upstream primer for the (lipase-2) gene is shown in SEQ ID No. 7, and the nucleotide sequence of the downstream primer is shown in SEQ ID No. 8. The nucleotide sequence of the upstream primer for the (liposynthetic enzyme-3) gene is shown in SEQ ID No. 9, and the nucleotide sequence of the downstream primer is shown in SEQ ID No. 10; the nucleotide sequence of the upstream primer for the (lipoprotein lipase) gene is shown in SEQ ID No. 11, and the nucleotide sequence of the downstream primer is shown in SEQ ID No. 12. The nucleotide sequence of the upstream primer for the (sterol regulatory element binding protein-1c-1) gene is shown in SEQ ID No. 13, and the nucleotide sequence of the downstream primer is shown in SEQ ID No. 14. The nucleotide sequence of the upstream primer for the (sterol regulatory element binding protein-1c-2) gene is shown in SEQ ID No. 15, and the nucleotide sequence of the downstream primer is shown in SEQ ID No. 16. The nucleotide sequence of the upstream primer for the (liver X receptor-α) gene is shown in SEQ ID No. 17, and the nucleotide sequence of the downstream primer is shown in SEQ ID No. 18. The nucleotide sequence of the upstream primer for the (liver X receptor-β) gene is shown in SEQ ID No. 19, and the nucleotide sequence of the downstream primer is shown in SEQ ID No. 20. The nucleotide sequence of the upstream primer of the (peroxisome proliferator-activated receptor-α) gene is shown in SEQ ID No. 21, and the nucleotide sequence of the downstream primer is shown in SEQ ID No. 22; The nucleotide sequence of the upstream primer for the (peroxisome proliferator-activated receptor γ-1) gene is shown in SEQ ID No. 23, and the nucleotide sequence of the downstream primer is shown in SEQ ID No. 24. The nucleotide sequence of the upstream primer of the (peroxisome proliferator-activated receptor γ-2) gene is shown in SEQ ID No. 25, and the nucleotide sequence of the downstream primer is shown in SEQ ID No. 26.

[0037] 7. Protein quantification and Western blot (immunoblotting) detection: Protein extraction: ① Take a small piece of tissue and place it in a pre-labeled 1.5mL centrifuge tube, then add... RIPA lysis buffer (containing protease inhibitors and phosphorylated protease inhibitors) ), grind and fully lyse using a tissue homogenizer; ② Centrifuge the sample at 13000×g, 4℃ for 5 min; ③ Take the supernatant as the protein sample (be careful not to aspirate the precipitate). The sample color is slightly light yellow.

[0038] Protein quantification and denaturation: ① Protein quantification was performed using the BCA Protein Assay kit (Beijing Kangwei Century Biotechnology Co., Ltd.). BSA (bovine serum albumin) standards were diluted in a concentration gradient (mass concentrations of 0, 62.5, 125, 250, 500, 1000, and 2000 ng / μL, respectively) and added to the protein sample to be tested (diluted 10 times) into labeled centrifuge tubes. ② Add to each pipe BCA (Bicinchoninic Acid) working solution: After shaking and mixing, incubate in a water bath at 37°C for 30 min, and then cool to room temperature. ③ Measure the absorbance of the sample and BSA standard at 562 nm using a spectrophotometer, plot the standard curve, and then calculate the protein concentration; ④ Take the supernatant Later added Boil at 100℃ for 10 minutes (to fully denature), and store at -20℃.

[0039] Western blot analysis: ① Gel preparation: Select the appropriate concentration of SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel) according to the molecular weight of the target protein, and prepare a 12% separating gel and a 5% stacking gel. ② Sample loading: Sample volume per well protein; ③ Electrophoresis: The entire SDS-PAGE process is performed under constant voltage. The stacking gel voltage is 80V and the time is 30min; the separating gel voltage is 120V and the time is 60min. Electrophoresis is stopped when the bromophenol blue reaches near the bottom of the gel. ④ Transfer and blocking: PVDF (polyvinylidene fluoride) membranes are activated with anhydrous methanol, and the membranes are selected according to protein size. Transfer membrane; soak all materials in transfer solution for 10 min, and clamp them in the following order: black side - sponge - 3 layers of filter paper - glue - PVDF membrane - 3 layers of filter paper - sponge - white side (avoid air bubbles), transfer membrane at constant current of 250mA for 2.5 h, and then seal with 5% skim milk powder (TBST) at room temperature for 2 h after transfer. ⑤ Immunoassay: After washing the membrane once with TBST, incubate in blocking buffer at room temperature for 1 hour; wash the membrane three times (10 min) with TBST on a shaker for decolorization; incubate overnight at 4°C with primary antibody (TBST diluted) (protein side down); then wash three times (10 min) with TBST; incubate with secondary antibody (TBST diluted) at room temperature for 1 hour; then wash three times (10 min) with TBST on a shaker for decolorization; and perform chemiluminescence reaction. ⑥ Chemiluminescence development: Place the membrane protein side up into the chemiluminescence imaging system, mix equal volumes of ECL (electrochemiluminescence solution) A / B in a 1.5mL EP tube, and drop it onto the membrane surface for exposure and development; ⑦ Band analysis: Image J 1.49p software was used for relative quantitative analysis of protein band gray values.

[0040] (III) Experimental Results: 1. Effects of scallion extract on body weight, food intake, and energy in rats of different groups: like Figure 1 As shown in Table 1, there was no significant difference in body weight among the groups before the experiment (P>0.05), and the body weight of the rats in each group continued to increase over time. At week 7, the body weight of rats in the HF and HFHL groups was significantly lower than that in the NC group (P<0.05); at week 12, the body weight of the HL group was significantly higher than that in the NC group (P<0.05). Compared with the HL group, the body weight of rats in the HL-AMR group was significantly lower (P<0.05). As shown in Table 2, in terms of dietary intake, the feed intake of the HF, HL, and HFHL groups was significantly lower than that of the normal control group (P<0.01), and there was no significant change between the scallion extract intervention groups and their corresponding model groups. In addition, the fructose intake of rats in the HF-AMR group was higher than that in the HF group, and the fructose intake of rats in the HFHL-AMR group was lower than that in the HFHL group. The energy intake of rats in the HF, HL, and HFHL groups was significantly higher than that in the NC group, and all of the above differences were statistically significant (P<0.05 or P<0.01), while there was no significant difference in energy intake between the scallion extract intervention groups and their corresponding model groups.

[0041] Table 1. Effects of scallion extract on the body weight of rats in each group. Note: Compared with the NC group, * P<0.05, ** P<0.01; compared with the HF group, # P<0.05, ## P<0.01; compared with the HL group, , .

[0042] Table 2. Effects of scallion extract on food intake and energy in rats of each group. Note: Compared with the NC group, *P<0.05, **P<0.01; compared with the HF group, # P<0.05, ## P<0.01; compared with the HFHL group, , .

[0043] 2. Effects of scallion extract on tissue weight in rats of different groups: As shown in Table 3, after 12 weeks of feeding, in terms of liver tissue: compared with the NC group, the liver wet weight of rats in the HL group increased significantly at all time points, and the liver coefficient of rats in the HL group also increased significantly at week 12. The liver coefficient of rats in the HFHL group increased at week 7. Compared with the HFHL group, the liver wet weight and liver coefficient of rats in the HFHL-AMR group increased significantly at week 12 (P<0.05 or P<0.01).

[0044] Table 3. Changes in liver tissue weight and corresponding indices of rats in each group ( ) Note: Compared with the NC group, *P<0.05, **P<0.01; compared with the HFHL group, , .

[0045] As shown in Table 4, regarding cardiac tissue: compared with the NC group, the heart mass and cardiac coefficient of rats in the HF and HFHL groups decreased at week 12, and the cardiac coefficient of the HL group decreased significantly; compared with the HF group, the cardiac coefficient of rats in the HF-AMR group increased significantly at week 12; compared with the HFHL group, the cardiac coefficient of rats in the HFHL-AMR group decreased significantly at week 12 (P<0.05 or P<0.01).

[0046] Table 4. Changes in heart tissue weight and corresponding indices of rats in each group ( ) Note: Compared with the NC group, *P<0.05, **P<0.01; compared with the HFHL group, , .

[0047] As shown in Table 5, regarding kidney tissue: compared with the NC group, the kidney coefficient of the HL group rats decreased at all time points; compared with the HL group, the kidney coefficient of the HL-AMR group rats increased at week 12; compared with the HFHL group, the kidney coefficient of the HFHL-AMR group rats also increased at week 12 (P<0.05 or P<0.01).

[0048] Table 5 Changes in kidney tissue weight and corresponding indices in each group of rats ( ) Note: Compared with the NC group, *P<0.05, **P<0.01; compared with the HF group, # P<0.05, ## P<0.01; compared with the HFHL group, , .

[0049] As shown in Table 6, regarding genital tissue: compared with the NC group, the genital mass of rats in the HF group, HL group, and HFHL group decreased at week 12 of the experiment, and the genital coefficient of the HL group also decreased; compared with the HF group, the genital mass and coefficient of rats in the HF-AMR group increased at week 12; compared with the HL group, the genital mass and coefficient of rats in the HL-AMR group were improved at week 12 (P<0.05 or P<0.01).

[0050] Table 6. Changes in the weight of genital tissues and their corresponding indices in each group ( ) Note: Compared with the NC group, *P<0.05, **P<0.01; compared with the HF group, # P<0.05, ## P<0.01; compared with the HL group, & P<0.05, && P<0.01; compared with the HFHL group, , .

[0051] 3. Wild onion extract lowers blood sugar, insulin, and HOMA-IR levels: like Figure 2 As shown, compared with the NC group, the serum blood glucose and insulin levels of rats in the HF, HL, and HFHL groups were significantly increased at each time point (P<0.05 or P<0.01). After adding scallion extract, compared with the HF group, the serum blood glucose and insulin levels of rats in the HF-AMR group were significantly decreased at each time point (P<0.05 or P<0.01); compared with the HL group, the serum blood glucose level of the HL-AMR group was significantly decreased at all time points, and the insulin level was significantly decreased at week 12 (P<0.05 or P<0.01); compared with the HFHL group, the serum blood glucose level of rats in the HFHL-AMR group was significantly decreased at all time points, and the insulin level was significantly decreased at week 12 (P<0.05 or P<0.01). The HOMA-IR index of rats in each group was calculated. The results showed that the HOMA-IR index of rats in the HF, HL and HFHL groups was significantly higher than that in the NC group at each observation time point (all P<0.01), indicating that the degree of insulin resistance was becoming more and more obvious. After intervention with scallion extract, at weeks 7 and 12, the scallion intervention groups were significantly lower than the corresponding model groups (all P<0.01).

[0052] 4. Sand onion extract reduces serum biochemical marker levels: like Figure 3As shown, at week 7, compared with the NC group, the serum TC and LDL-C levels of rats in the HF group were significantly increased (P<0.05 or P<0.01); the serum TG and LDL-C levels of rats in the HL group were significantly increased, while the serum HDL-C level was significantly decreased (both P<0.01); the serum TC level of rats in the HFHL group was significantly increased (P<0.01). After intervention with scallion extract, compared with the HF group, the LDL-C level of the HF-AMR group was significantly decreased, while the HDL-C level was increased (both P<0.01); compared with the HL group, the TG and LDL-C levels of the HL-AMR group were significantly decreased, while the HDL-C level was increased (both P<0.01); compared with the HFHL group, the TC level of the HFHL-AMR group was decreased (P<0.05).

[0053] like Figure 4 As shown, at week 12, compared with the NC group, serum TC, TG, and LDL-C levels were significantly increased in the HF, HL, and HFHL groups, while HDL-C concentration was significantly decreased in the HF, HL, and HFHL groups (all P < 0.01). After intervention with scallion extract, serum TC, TG, and LDL-C levels in all groups were significantly decreased (all P < 0.05 or P < 0.01). Except for the HFHL group, where serum HDL-C levels remained unchanged, the increases in serum HDL-C levels in the other groups were statistically significant (P < 0.05 or P < 0.01).

[0054] 5. Shallot extract improves fatty liver degeneration: After feeding rats a high-fructose, high-fat diet for 12 weeks, the appearance and morphology of the rat liver were observed by visual inspection. Figure 5 As shown, at a magnification of 200× and a scale of [missing information], In the NC group, the livers of rats appeared normal, reddish-brown, with a smooth surface and sharp edges. The livers of rats in the HF group were darker in color than those in the NC group, with decreased luster, softer texture, and a slightly oily feel. Compared with the NC group, the livers of rats in the HL group were lighter in color, yellowish-brown, significantly larger in volume, with a more pronounced oily texture, blunted edges, and visible fat granules. The livers of rats in the HFHL group were darker in color than those in the NC group, moderately larger in volume, with slightly blunted edges and an oily feel on the cut surface. The livers of rats in each of the scallion extract intervention groups showed varying degrees of improvement.

[0055] The results of hematoxylin-eosin (HE) staining of the liver are as follows: Figure 6As shown, at each time point, the liver cells of rats in the NC group were normal, with clear and normal structure, and no abnormalities were found in liver pathological examination. In the HF group, the fatty degeneration of liver cells worsened over time, and liver cell vacuolation was obvious. In the HL group, the degree of fatty degeneration of liver tissue gradually deteriorated with the extension of modeling time, with disordered liver cell structure, unclear boundaries, a large number of lipid droplets and cell ballooning degeneration, and obvious inflammatory reactions and other pathological changes. In the HFHL group, disordered cell arrangement, extensive and obvious vacuolation, and severe fatty degeneration were observed at all time points. However, under the intervention of sand onion extract, the fatty degeneration of liver in rats in all groups was significantly improved, and the liver structure was restored to varying degrees.

[0056] 6. The extract of *Allium tuberosum* reduces the mRNA expression levels of genes related to liver fat metabolism: Real-time PCR test results as follows Figure 7 As shown, at week 7, compared with the NC group, the liver tissue of rats in the HF group... , , , Expression increased significantly. , Expression was significantly decreased; liver tissue of HL group rats FAS , , , Expression was significantly increased in liver tissue of HFHL group rats. , Expression increased significantly. Expression was significantly reduced (P<0.05 or P<0.01). Intervention with *Allium tuberosum* extract significantly upregulated liver tissue expression in the NC group rats. The expression was lowered. , , The expression of [the substance] was significantly upregulated in the HF group rats; HSL , LPL , The expression was suppressed. , , Expression; In HL-AMR group rats, scallion extract was found to... , , , The levels were significantly downregulated; meanwhile, the extract intervention significantly reduced the levels in rats fed a high-fructose, high-fat diet. The level was significantly improved (P<0.05 or P<0.01).

[0057] like Figure 8 As shown, at week 12, compared with the NC group, the liver tissue of rats in the HF group, HL group, and HFHL group... , , Expression was significantly upregulated in all cases, including LPL. Expression was significantly downregulated in both the HL and HFHL groups; in addition, expression was significantly downregulated in both groups. The expression was significantly improved in the HL group. Expression was significantly downregulated (P<0.05 or P<0.01). Intervention with scallion extract reduced expression in all groups of rats. , , The expression has been expanded to include HSL, The expression of [something] was also upregulated by scallion extract in rats fed high-fructose and high-fructose-high-fat diets. The expression of [specific substance] was downregulated in rats exposed to a high-fat diet and a combined high-fructose high-fat diet. The expression of HSL in the liver tissue of rats in the normal diet group was found to be negative (P<0.05 or P<0.01); in addition, HSL was also found in the liver tissue of rats in the normal diet group. The expression is adjusted upwards. Expression was downregulated (all P < 0.01).

[0058] Western Blot results are as follows Figure 9 As shown, in week 7, compared with the NC group, the HF group, HL group, and HFHL group... , Protein levels were significantly increased in the HF group. , Protein levels increased significantly, while HSL and LPL protein levels decreased significantly; LPL protein levels decreased somewhat in the HL group; HFHL group , Protein levels decreased (P<0.05 or P<0.01). After intervention with sand onion extract, Group Protein expression levels were downregulated compared to the NC group; HSL and LPL protein expression increased in the HSL group compared to the HF group, while FAS and LPL protein expression increased in the HF group. Protein levels were lower than in the HF group; Group Protein expression levels were lower than in the HL group; Group Protein levels were higher than in the HFHL group (P<0.05 or P<0.01).

[0059] The results for week 12 are as follows: Figure 10 As shown, compared with the NC group, rats in each dietary exposure group... Protein expression levels were significantly increased. Protein expression was significantly reduced in all groups; in addition, the HF group , Protein expression was upregulated, and HSL and LPL protein levels were decreased; HL group Increased protein levels, with a significant decrease in HSL protein levels; FAS in the HFHL group, , Protein expression was significantly upregulated (P<0.05 or P<0.01). After intervention with scallion extract, the expression of HSL protein in the liver tissue of rats on a normal diet was increased, while the expression of FAS protein was decreased; the intervention also increased LPL expression in the high-fructose group. Increased protein expression, FAS, Protein levels decreased; HSL levels increased in high-fat diet rats. Protein levels decreased FAS, , , Protein levels; also reduced HSL in rats fed a high-fructose, high-fat diet. Protein expression also increased, FAS, Protein levels were significantly reduced (P<0.05 or P<0.01).

[0060] In summary, the *Allium tuberosum* extract of this invention can be used to prepare a diet to improve metabolic-related fatty liver disease. Compared with the NC group, the HL group rats showed significantly increased body weight, liver wet weight, and liver coefficient. *Allium tuberosum* significantly reduced the body weight of rats in the high-fat diet group and effectively reduced blood glucose and insulin levels. The HF, HL, and HFHL groups of rats in this invention... The indices were all significantly higher than those of the normal control group, indicating that a high-fat diet and fructose exposure in early life can lead to insulin resistance (IR) in rats. When each model group was treated with scallion extract, the IR was significantly reduced. The levels of all these substances were significantly reduced, effectively improving hyperinsulinemia in rats. It also effectively reduced serum biochemical levels. After intervention with scallion extract, except for no change in serum TC levels under a high-fructose diet and serum HDL-C levels under a high-fructose combined with a high-fat diet, serum TC, TG, and LDL-C levels were reduced under all other dietary conditions. Histological observation showed that intervention with scallion extract could reverse the increase in lipid deposition and hepatocyte vacuolation in rat livers, suggesting that scallion extract may correct lipid metabolism disorders by improving IR in rats and has a potential intervention effect on MAFLD, and can be used to improve hepatic steatosis. Scallion extract intervention could downregulate the expression levels of FAS protein and gene in the liver tissue of rats in the high-fructose group. The expression of [something] caused [something] in the liver tissue of rats in the high-fat diet group. , The expression of FAS protein and gene was reduced to varying degrees, resulting in decreased liver tissue expression in rats fed a high-fructose and high-fat diet. , , FAS protein expression was downregulated.

[0061] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. An application of a sand onion extract, characterized in that, The scallion extract is used to prepare feed to improve metabolic-related fatty liver disease.

2. The application of the sand onion extract according to claim 1, characterized in that, The application includes one or more of the following: The sand onion extract is used to lower serum blood glucose levels; The sand onion extract is used to lower serum insulin levels; The sand onion extract is used to reduce serum biochemical levels. The scallion extract is used to improve hepatic steatosis; The scallion extract is used to reduce the mRNA expression level of genes related to liver fat metabolism.

3. The application of the sand onion extract according to claim 2, characterized in that, When the scallion extract is used to lower serum blood glucose and serum insulin levels, it can significantly reduce the serum insulin resistance index.

4. The application of the sand onion extract according to claim 2, characterized in that, When the sand onion extract is used to reduce serum biochemical levels, the serum biochemical levels include TC, TG, and One or more.

5. The application of the sand onion extract according to claim 2, characterized in that, When the scallion extract is used to reduce the mRNA expression level of liver lipid metabolism-related genes, the liver lipid metabolism-related genes include , , and One or more.

Images