Use of total saponins extract of morinda officinalis how in preparation of medicine for treating MAFLD

Abstract

This invention belongs to the field of traditional Chinese medicine and discloses the application of total saponins extract of *Abrus precatorius* in the preparation of drugs for treating metabolic-associated fatty liver disease (MAFLD). Through experiments in high-fat diet-induced MAFLD mice, this invention demonstrates that total saponins of *Abrus precatorius* can protect the liver from a series of damages caused by excessive lipid accumulation. The potential mechanisms include regulating lipid metabolism through the PPAR and AMPK signaling pathways, and modulating the balance of gut microbiota. These findings provide a solid research foundation and empirical evidence for the potential application of total saponins of *Abrus precatorius* in the treatment of MAFLD.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine, specifically relating to the application of a total saponin extract of *Abrus precatorius* in the preparation of a drug for treating metabolic-associated fatty liver disease (MAFLD). Background Technology

[0002] Metabolic dysfunction-associated fatty liver disease (MAFLD) is a chronic, progressive liver disease caused by overnutrition and insulin resistance in genetically susceptible individuals. The disease spectrum includes metabolic-associated fatty liver, metabolic-associated steatohepatitis, and related fibrosis and cirrhosis. Currently, clinical intervention for MAFLD mainly relies on lifestyle modifications (including dietary adjustments, exercise, and weight management) and drug therapy, but the effects are limited, and some drugs may cause side effects.

[0003] Currently, domestic treatments for metabolic-associated fatty liver disease (MAFLD) primarily focus on improving metabolic disorders, protecting hepatocytes, and reducing inflammation and fibrosis. There are no specifically approved drugs for this condition, and clinical use is often off-label or combined with treatment for comorbidities such as metabolic syndrome (obesity, diabetes, hyperlipidemia). Internationally, the US FDA approved Resmetirom in 2024 and Semaglutide in 2025 for adult patients without cirrhosis and with moderate to severe liver fibrosis (F2-F3 stages). However, Resmetirom is not yet available in China, and Semaglutide has drawbacks such as numerous gastrointestinal side effects. Furthermore, both drugs primarily target patients with advanced MAFLD, and there are no effective intervention strategies to block early disease progression. Therefore, there is an urgent need to develop new drugs for MAFLD to address this serious public health problem in China.

[0004] *Abrus cantoniensis*, the dried whole plant of a legume, is a commonly used medicinal herb in the Lingnan region and is listed in the Chinese Pharmacopoeia (2020 edition). It has a sweet and slightly bitter taste, and is cool in nature. It possesses properties of clearing heat and detoxifying, promoting diuresis and relieving jaundice, and soothing the liver and relieving pain. It is often used to treat damp-heat jaundice, discomfort in the hypochondrium, abdominal distension and pain, and mastitis. Clinically, compound preparations based on *Abrus cantoniensis* have been widely used to treat hepatitis and liver fibrosis, showing certain efficacy and suggesting its significant potential in treating liver diseases. However, its main active ingredients and specific mechanisms of action remain unclear. Further research is needed on the active components and specific molecular mechanisms of *Abrus cantoniensis* in treating MAFLD to lay the foundation for developing new drugs for MAFLD. Summary of the Invention

[0005] The purpose of this invention is to overcome at least one deficiency of the prior art and to provide the use of total saponin extract of *Abrus precatorius* in the preparation of a medicament for the treatment of metabolic-associated fatty liver disease (MAFLD).

[0006] The technical solution adopted in this invention is:

[0007] In a first aspect, the present invention provides the use of total saponin extract of *Abrus precatorius* in the preparation of a medicament for treating metabolic-related fatty liver disease.

[0008] In some embodiments, the total saponin extract of *Abrus precatorius* provided by the present invention can be prepared by the following method:

[0009] 1) After coarsely crushing the chicken bone grass, extract it using the hot reflux method with ethanol. After concentrating the extract, obtain the ethanol concentrate, and add water to obtain the crude extract A.

[0010] 2) Petroleum ether was added to crude extract A for extraction, and the aqueous layer was collected to obtain crude extract B;

[0011] 3) Add n-butanol to crude extract B for extraction, collect the n-butanol layer, evaporate the n-butanol to dryness, and dissolve it in water to obtain crude extract C;

[0012] 4) The crude extract C was purified by passing it through a macroporous resin, eluted with water and ethanol, the eluent was collected, the ethanol was recovered, and the extract was concentrated. In some embodiments, the total saponin extract of *Abrus precatorius* contains daidzein I, sophora japonica saponin III, dehydrodaidzein I, absinthecin F and absinthecin SO1.

[0013] In some embodiments, the adult dosage of the total saponin extract of *Abrus precatorius* is 50-300 mg daily.

[0014] In some embodiments, the medicament comprises total saponin extract of *Abrus precatorius* and pharmaceutically acceptable carriers or excipients.

[0015] In some embodiments, the dosage form of the drug is an oral liquid, pill, granule, capsule, tablet, drop pill, injection, or powder.

[0016] In some embodiments, the total saponin extract of *Abrus precatorius* is selected from petroleum ether extract, ethyl acetate extract, n-butanol extract and / or water extract of total saponins of *Abrus precatorius*.

[0017] In some embodiments, the total saponin extract of *Abrus precatorius* can regulate gene expression related to lipid synthesis and metabolism, fatty acid oxidation metabolism, and lipid transport and binding.

[0018] In some embodiments, the total saponin extract of *Abrus precatorius* can improve liver fibrosis levels.

[0019] In some embodiments, the total saponin extract of *Abrus precatorius* can improve liver inflammation levels.

[0020] The beneficial effects of this invention are:

[0021] In MAFLD mice induced by a high-fat diet, treatment with total saponins from *Abrus precatorius* significantly reduced body weight, liver index, hepatic steatosis, oxidative stress, inflammation, and fibrosis, thereby improving liver pathology and reducing NAS scores.

[0022] Network pharmacology analysis predicted that total saponins from *Abrus precatorius* would improve MAFLD by regulating the PPAR and AMPK signaling pathways. Western blot analysis confirmed that total saponins from *Abrus precatorius* activated AMPK, inhibited SREBF1 expression, and promoted phosphorylation and inactivation of ACC, thereby reducing lipid synthesis. Simultaneously, total saponins from *Abrus precatorius* activated PPARα, promoted CPT1A expression, and accelerated lipid oxidation. Furthermore, treatment with total saponins from *Abrus precatorius* restored the gut microbiota imbalance induced by a high-fat diet, reducing the abundance of harmful bacteria and increasing the abundance of beneficial bacteria.

[0023] In summary, the high-fat diet-induced MAFLD mouse experiment demonstrated that total saponins from *Abrus precatorius* can protect the liver from a range of damage caused by excessive lipid accumulation. The potential mechanisms include regulating lipid metabolism through the PPAR and AMPK signaling pathways, as well as modulating the balance of gut microbiota. These findings provide a solid research foundation and empirical evidence for the potential application of total saponins from *Abrus precatorius* in the treatment of MAFLD. Attached Figure Description

[0024] Figure 1 This is the chemical fingerprint of the total saponins sample from *Abrus precatorius*.

[0025] Figure 2 The data are expressed as Mean ± SD (n=8). Compared with the NC-Vehicle group, * < 0.05, ** < 0.01, *** < 0.001; compared with the HFD-Pioglitazone group, #p < 0.05, ##p < 0.01, ###p < 0.001; #p < 0.05, ##p < 0.01, ###p < 0.001; compared with the HFD-ACS-L group, #l < 0.05, ##l < 0.01, ###l < 0.001; compared with the HFD-ACS-H group, #h < 0.05, ##h < 0.01, ###h < 0.001.

[0026] Figure 3The graph shows the changes in liver weight and liver index (the ratio of liver weight to body weight) in mice (n=8).

[0027] Figure 4 H&E stained sections of mouse liver and NAS pathological scoring images (n=5).

[0028] Figure 5 Images showing the TG and TC content in mouse livers and Oil Red O staining of liver tissue sections (n=5).

[0029] Figure 6 The figure shows the effects of total saponins from *Abrus precatorius* on liver function and lipid metabolism in HFD-induced MAFLD mice (n=5). Figure A shows the serum ALT and AST levels of mice, and Figure B shows the serum glycolipid levels of mice.

[0030] Figure 7 The study investigated the regulation of lipid metabolism-related genes in HFD-induced MAFLD mice by total saponins of *Abrus precatorius* (n=5). Figure A shows genes related to lipid synthesis and metabolism; Figure B shows genes related to lipid oxidation metabolism; and Figure C shows genes related to lipid transport and binding.

[0031] Figure 8 The study investigated the effects of total saponins from *Abrus precatorius* on the level of liver fibrosis in HFD-induced MAFLD mice (n=5). Figure A shows Sirius red staining (200×) on mouse liver tissue; Figure B shows the mRNA expression level in mouse liver fibrosis.

[0032] Figure 9 The effect of total saponins from *Abrus precatorius* on the level of liver oxidative stress in HFD-induced MAFLD mice (n=5).

[0033] Figure 10 The effect of total saponins from *Abrus precatorius* on the level of inflammatory cell infiltration in the liver tissue of HFD-induced MAFLD mice (n=5) is shown in Figure A, where F4 / 80 immunohistochemical staining of mouse liver tissue (200×) is shown; and F4 / 80 positive cell rate is shown in Figure B.

[0034] Figure 11 The effect of total saponins from *Abrus precatorius* on the expression level of liver inflammatory factors mRNA in HFD-induced MAFLD mice (n=5).

[0035] Figure 12 The effects of total saponins from *Abrus precatorius* on the expression of proteins in the AMPK / SREBF1 / ACC and PPARα / CPT1A pathways induced by HFD in MAFLD mice (n=5) are shown in Figure A, which represents the phosphorylation levels of AMPK, ACC, and SREBF1; and the protein levels of PPARα and CPT1A. Detailed Implementation

[0036] The following disclosure provides many different implementations or examples for different ways of implementing the present invention.

[0037] It should be noted that, in the appendix to this application Figure 3 To be continued Figure 12 In the figures, all data are expressed as Mean ± SD, and the symbols have the following meanings: Compared with the NC-Vehicle group, *P < 0.05, **P < 0.01, ***P < 0.001; Compared with the HFD-Vehicle group, # P < 0.05 ## P < 0.01, ### P < 0.001. Unless otherwise stated, this will not be repeated below.

[0038] Example

[0039] 1. Preparation and purification process of total saponins from *Abrus precatorius*

[0040] The preparation and purification process of total saponins from *Abrus precatorius* in this invention can refer to Chapter 2 of the prior study "Yao Xiangcao. Extraction process and anti-HBV activity and mechanism of action of total saponins from *Abrus precatorius* [D]. Guangdong: Southern Medical University, 2019."

[0041] LC-MS analysis showed that the saponin content of the sample obtained by this process reached 61.25%.

[0042] 2. Isolation and Identification of Major Saponin Monomers

[0043] The extraction steps are as follows:

[0044] To clarify and extract the effective components of total saponins, the total saponin sample from *Abrus precatorius* obtained in the previous section was subjected to silica gel column chromatography with gradient elution using a dichloromethane / methanol system, yielding four fractions (Fr. 1–4). Fr. 4 was then subjected to Sephadex LH-20 gel column chromatography with isocratic elution using dichloromethane / methanol (1:1, v / v), yielding six subfractions (Fr. 4.1–4.6).

[0045] Fr. 4.2 was subjected to silica gel column chromatography with a gradient elution of dichloromethane / methanol / 0.1% formic acid-water system to obtain 5 subfractions (Fr. 4.2.1~4.2.5). Finally, Fr. 4.2.4 was purified by ODS column chromatography and high performance liquid chromatography (HPLC) to obtain 5 target compounds: soyasaponin I, Huaihuasaponin III, dehydrosoyasaponin I, abrucantoside F, and abrucantoside SO1.

[0046] The analysis is as follows:

[0047] UPLC-TOF analysis revealed that the total saponin sample extracted in the previous section contained 25 saponin components. After purification by column chromatography and high performance liquid chromatography, the contents of five saponin components, namely absinin F, daidzein I, dehydro-daidzein I, sophorae glycoside III, and absinin SO1, were relatively high.

[0048] Subsequently, using absinthecein F, daidzein I, dehydrodaidzein I, sophoraesodium saponin III, and absinthecein SO1 as the detection targets, a chemical fingerprint of the total saponin sample was established. First, mixed reference solutions of different concentrations of the five active ingredients and the sample test solution were prepared. Appropriate chromatographic conditions were selected, and HPLC was used for detection. The elution times of the five components were determined, and the peak areas of the five characteristic peaks were set to be greater than 50% of the total peak area, serving as the chemical fingerprint of the total saponins from *Abrus precatorius* and the extraction quality standard (e.g., ...). Figure 1 As shown, peak 1: soybean saponin I; peak 2: sophora saponin III; peak 3: dehydro-soybenoside I; peak 4: absinin SO1; peak 5: absinin F).

[0049] 3. Establishment of animal models and related experimental analysis

[0050] 3.1 Experimental methods and procedures

[0051] First, the solution preparation method is as follows:

[0052] 1) The method for preparing 0.5% CMC-Na (sodium carboxymethyl cellulose) solution is as follows: Weigh 0.25g of CMC-Na into 50mL of ultrapure water and sonicate in a 70℃ water bath until dissolved.

[0053] 2) Weigh 200 mg of total saponins from *Abrus precatorius* powder and dissolve it in 1 mL of DMSO. Then add 1 mL of 0.5% CMC-Na solution and sonicate to dissolve, thus obtaining the total saponins mother liquor from *Abrus precatorius* (100 mg / mL).

[0054] 3) Pioglitazone tablets can be dissolved in the same solvent as the total saponins of *Heliotropium indicum* in 0.5% CMC-Na solution to prepare the desired concentration.

[0055] Then, the experimental modeling method is as follows:

[0056] C57BL / 6J mice were selected and fed a high-fat diet (HFD) for 20 weeks to establish a MAFLD model. After successful model establishment, drug intervention was performed for 8 weeks.

[0057] The experimental groups and drug dosages are as follows:

[0058] The Chinese Pharmacopoeia recommends an adult dosage of 10-30g of *Abrus precatorius*. Using twice the maximum effective adult dose of 60g, and applying the Meeh-Rubner formula, the mouse dosage is calculated to be 9.1 times that of humans. Given that the crude drug ratio of total saponins in *Abrus precatorius* is 8.44 mg / 1 g crude drug, the calculated mouse dosage is 77 mg / kg. Therefore, this animal experiment designed low and high dose groups of total saponins from *Abrus precatorius* at 50 mg / kg and 100 mg / kg, respectively. The positive control group received pioglitazone, currently recommended in clinical guidelines for treating MAFLD, with a typical dosage of 10 mg / kg in mouse models.

[0059] The groups were as follows: normal diet (NC) control group, solvent (0.5% CMC-Na) control group (HFD-Vehicle), positive control group given pioglitazone (HFD-Pioglitazone) 10 mg / kg, low-dose group (HFD-ACS-L) given total saponins of *Abrus precatorius* 50 mg / kg, and high-dose group (HFD-ACS-H) given total saponins of *Abrus precatorius* 100 mg / kg.

[0060] After the experiment, serum, liver tissue and fecal samples were collected for subsequent phenotypic analysis, pathological examination, biochemical index detection, gene expression analysis and 16S rRNA sequencing.

[0061] 3.2 Analysis of mouse body weight, liver weight, and liver index

[0062] The changes in mouse body weight during the experiment are as follows: Figure 2 As shown, compared with the NC diet, the body weight of mice in the HFD diet group was significantly increased (P < 0.001). After treatment with pioglitazone, low dose (50 mg / kg) and high dose (100 mg / kg) of total saponins of *Abrus precatorius*, the weight gain trend of mice was significantly reduced compared with the solvent group (P < 0.001).

[0063] The changes in liver weight and liver index (liver weight to body weight) in mice are as follows: Figure 3As shown, compared with the NC-Vehicle group, the liver weight and liver index of mice in the HFD-Vehicle group were significantly increased (P<0.001, P<0.05). After treatment with pioglitazone, low dose (50mg / kg) and high dose (100mg / kg) of total saponins of *Abrus precatorius*, the liver weight and liver index of mice in the HFD-Vehicle group were significantly reduced compared with the solvent group (P<0.001).

[0064] 3.3 Analysis of histopathological characteristics of liver tissue

[0065] The NAS scoring criteria for liver histopathological feature analysis are as follows:

[0066] Five fields of view were randomly acquired from the liver H&E stained sections of mice in each group at 200× magnification. According to the MAFLD Activity Score (NAS) developed by the Pathology Committee of the NASH Clinical Research Network of the National Institutes of Health, which is recommended by the "Consensus on Diagnosis and Treatment of Nonalcoholic Fatty Liver Disease in the Asia-Pacific Region", three indicators of steatosis, ballooning degeneration and lobular inflammation in the field of view were scored. The scores of each indicator were summed to form the NAS score, which was used to evaluate the degree of MAFLD lesions in the liver. The scoring criteria are shown in Table 3-1.

[0067] Table 3-1 NAS Scoring Criteria

[0068] Table 3-1 NAS histological score

[0069]

[0070] H&E staining of mouse liver sections as shown Figure 4 As shown in Figure A, the normal control group mice showed uniform hepatocyte arrangement, intact liver lobules, and regular radial hepatic cords. The HFD-Vehicle group mice showed abnormal liver lobule structure, disordered hepatocyte arrangement, numerous fat vacuoles and ballooning degeneration, and obvious inflammatory cell infiltration in the liver sinusoids. Both pioglitazone and total saponins of *Abrus precatorius* showed significant improvement in pathological features, with relatively neat hepatocyte arrangement, only a few fat vacuoles and ballooning degeneration, and improved inflammatory cell infiltration. Furthermore, the high-dose (100 mg / kg) total saponins of *Abrus precatorius* showed a better improvement effect than the positive control drug pioglitazone.

[0071] Five fields of view were randomly selected from the H&E-stained liver sections of mice in each group for NAS pathological scoring. The results are as follows: Figure 4As shown in Figure B, the normal control group mice had a score of 0 for fatty degeneration, ballooning degeneration, lobular inflammation, and total NAS. In the HFD-Vehicle group, the scores for fatty degeneration, ballooning degeneration, and lobular inflammation were 1.98, 1.80, and 2.01, respectively, with a total NAS score > 5, indicating that the model group exhibited significant MAFLD characteristics. In the high-dose (100 mg / kg) *Abrus precatorius* total saponins administration group, the scores for fatty degeneration, ballooning degeneration, and lobular inflammation were 0.40, 0.42, and 1.13, respectively, with a total NAS score improvement exceeding 2 points. This indicates that *Abrus precatorius* total saponins significantly improved the pathological characteristics of MAFLD (P < 0.001), and the effect was superior to the positive control drug pioglitazone.

[0072] 3.4 Analysis of hepatic lipid accumulation in mice

[0073] Lipid accumulation in mouse liver was evaluated by detecting TG and TC levels and staining liver tissue sections with Oil Red O. The results of Oil Red O staining are shown below. Figure 5 As shown in Figure A, numerous red lipid droplets were deposited in the liver tissue of mice in the HFD-Vehicle group. After treatment with different doses of total saponins from *Abrus precatorius*, the number and volume of lipid droplets in the liver tissue of mice were reduced, with the high-dose (100 mg / kg) group showing the most significant improvement. The experimental results are as follows... Figure 5 As shown in Figure B, compared with the normal control group, the TG and TC contents in the liver tissue of mice in the HFD-Vehicle group were significantly increased (P < 0.001). Compared with the HFD-Vehicle group, the TG and TC contents in the liver tissue of mice treated with different doses of total saponins of *Abrus precatorius* were significantly decreased (P < 0.001). These experimental results indicate that total saponins of *Abrus precatorius* can improve lipid accumulation in the liver of MAFLD mice.

[0074] 3.5 Liver function and lipid metabolism analysis

[0075] Liver function in mice was evaluated by detecting serum ALT and AST levels. The experimental results are as follows: Figure 6 As shown in Figure A, serum ALT and AST levels in the HFD-Vehicle group mice were significantly higher than those in the normal control group (P < 0.05, P < 0.001), indicating liver function impairment. After treatment with different doses of total saponins from *Abrus precatorius*, serum ALT and AST levels significantly decreased (P < 0.001), indicating that total saponins from *Abrus precatorius* exerted a liver-protective effect. The lipid metabolism of the mice is shown in Figure A. Figure 6As shown in Figure B, serum TG, TC, and LDL-C levels in the HFD-Vehicle group were significantly higher than those in the normal control group (P < 0.001). After treatment with different doses of total saponins from *Abrus precatorius*, serum TG, TC, and LDL-C levels all significantly decreased (P < 0.05, P < 0.001), indicating that total saponins from *Abrus precatorius* can regulate lipid metabolism in MAFLD mice. Furthermore, serum glucose levels in the HFD-Vehicle group were significantly elevated (P < 0.001). After treatment with different doses of total saponins from *Abrus precatorius*, serum glucose levels showed a decreasing trend. Although there was no significant difference in the low-dose group, the high-dose group significantly reduced serum glucose levels (P < 0.05).

[0076] 3.6 Analysis of lipid metabolism-related genes

[0077] The expression of lipid metabolism-related genes in the liver tissue of mice in each group was detected by real-time quantitative PCR. The experimental results are as follows: Figure 7 As shown.

[0078] Compared with the normal control group, the expression of lipid synthesis and metabolism-related genes Srebf1 (cholesterol regulatory element binding protein 1), Fasn (fatty acid synthase), Pparg (peroxisome proliferator-activated receptor γ), Acc (acetyl-CoA carboxylase), Scd1 (stearoyl-CoA desaturase 1), and Hmgcr (3-hydroxy-3-methylglutaryl-CoA reductase) in the liver of HFD-Vehicle mice was significantly increased (P < 0.001), suggesting a lipid metabolism imbalance in MAFLD mice.

[0079] Compared with the HFD-Vehicle group, the expression of the above genes was significantly downregulated after treatment with total saponins of *Abrus precatorius*, especially under high-dose treatment (P < 0.001), indicating that treatment with total saponins of *Abrus precatorius* can improve lipid synthesis and metabolic disorders. Figure 7 A). Simultaneously, treatment with different doses of total saponins from *Abrus precatorius* significantly upregulated the expression of lipid oxidation metabolism-related genes PPARα (peroxisome proliferator-activated receptor α), Acox1 (acyl-CoA oxidase 1), and Cpt1a (carnitine palmitoyltransferase 1A) (P < 0.05, P < 0.001). This upregulation indicates that total saponins from *Abrus precatorius* can promote fatty acid oxidation and reduce hepatic fat accumulation. Figure 7 B).

[0080] In addition, the treatment with total saponins of *Abrus precatorius* also regulated the expression of genes related to lipid transport and binding, significantly upregulating Apob (apolipoprotein B) and downregulating Fapp1 (fatty acid binding protein 1) expression (P < 0.05, P < 0.001). This indicates that total saponins of *Abrus precatorius* can also regulate lipid metabolism by affecting lipid transport and fatty acid binding, thereby further reducing fat accumulation in the liver. Figure 7 C).

[0081] The above experimental results indicate that total saponins from *Abrus precatorius* can improve lipid metabolism disorders and lipid accumulation in HFD-induced MAFLD mice by regulating the expression of genes related to lipid synthesis and metabolism, fatty acid oxidation metabolism, and lipid transport and binding.

[0082] 3.6 Analysis of the progression of liver fibrosis

[0083] The level of liver fibrosis in mice was assessed by Sirius red staining, and the results were as follows: Figure 8 As shown in Figure A, the liver tissue of mice in the HFD-Vehicle group showed obvious fibrotic lesions compared with the normal control group, and the amount of red collagen fibers was significantly increased. After treatment with different doses of total saponins of *Abrus precatorius*, the deposition of collagen fibers was reduced. The improvement effect of the high-dose (100 mg / kg) total saponins of *Abrus precatorius* group was better than that of the low-dose (50 mg / kg) group, and comparable to that of the positive control drug pioglitazone.

[0084] Hepatic stellate cells (HSCs) are activated by mediators such as inflammatory factors, inducing the production and deposition of large amounts of extracellular matrix (ECM), thereby driving the development of liver fibrosis. Transforming growth factor β (TGF-β), α-smooth muscle actin (α-SMA), and type I collagen A1 (COL1A1) are biomarkers of HSC activation. Therefore, the level of liver fibrosis in each group of mice was evaluated by detecting the mRNA expression levels of these three biomarkers. The experimental results are as follows: Figure 8 As shown in Figure B, the expression levels of Tgfb, Col1a1, and Acta2 genes in the liver tissue of mice in the HFD-Vehicle group were significantly higher than those in the normal control group (P < 0.001). Low-dose (50 mg / kg) total saponins of *Abrus precatorius* did not significantly reduce Tgfb gene expression, but it significantly reduced Col1a1 and Acta2 gene expression levels (P < 0.05). High-dose (100 mg / kg) total saponins of *Abrus precatorius* significantly reduced the expression levels of Tgfb, Col1a1, and Acta2 genes (P < 0.001). These experimental results indicate that total saponins of *Abrus precatorius* can significantly improve the level of liver fibrosis in HFD-induced MAFLD mice.

[0085] 3.7 Analysis of Oxidative Stress and Inflammation Levels

[0086] The oxidative stress levels of mice in each group were evaluated by detecting the levels of MDA and SOD in the liver. The experimental results are as follows: Figure 9 As shown, compared with the normal control group, the MDA level in the liver tissue of mice in the HFD-Vehicle group was significantly increased (P < 0.001), and the SOD level was significantly decreased (P < 0.001), indicating that the liver of HFD-induced MAFLD mice has a high level of oxidative stress. Compared with the HFD-Vehicle group, the MDA level in the liver tissue of mice treated with different doses of total saponins of *Abrus precatorius* was significantly decreased (P < 0.001). The SOD level showed a tendency to rise after low-dose treatment with total saponins of *Abrus precatorius*, but the difference was not significant. However, high-dose treatment with total saponins of *Abrus precatorius* significantly increased the SOD level in the liver tissue of mice (P < 0.05). These experimental results indicate that total saponins of *Abrus precatorius* can significantly improve oxidative stress in HFD-induced MAFLD mice.

[0087] F4 / 80 is a widely studied and cited membrane protein and a marker of mature mouse macrophages. Immunohistochemical staining of mouse liver tissue sections with F4 / 80 was performed, and the F4 / 80 positivity rate was calculated using the IHC Profiler plugin of ImageJ software to evaluate macrophage infiltration in mouse liver tissue. Experimental results are as follows: Figure 10 As shown, compared with the normal control group, the F4 / 80 positivity rate in the HFD-Vehicle group was significantly increased (P < 0.001), indicating that the macrophage infiltration in the liver of HFD-induced MAFLD mice was significantly enhanced. Compared with the HFD-Vehicle group, after treatment with different doses of total saponins from *Abrus precatorius*, the F4 / 80 positivity rate in the liver tissue of mice was significantly reduced (P < 0.01, P < 0.001), indicating that total saponins from *Abrus precatorius* can significantly improve inflammatory cell infiltration in the liver of HFD-induced MAFLD mice.

[0088] The inflammation levels of mice in each group were evaluated by detecting the gene expression of inflammatory factors in mouse liver tissue. The experimental results are as follows: Figure 11 As shown, compared with the normal control group, the expression levels of Tnfα, Il-1b and Il-6 genes in the liver tissue of HFD-Vehicle group mice were significantly increased (P < 0.001), while treatment with different doses of total saponins of *Abrus precatorius* significantly reduced the expression levels of Tnfα, Il-1b and Il-6 genes (P < 0.05, P < 0.001), indicating that total saponins of *Abrus precatorius* can significantly improve the inflammation level of liver in HFD-induced MAFLD mice.

[0089] 3.8 Regulatory Analysis of the AMPK / SREBF1 / ACC and PPARα / CPT1A Pathways

[0090] Based on network pharmacology analysis, this study predicted that total saponins from *Abrus precatorius* might improve MAFLD through the AMPK and PPAR signaling pathways. This study verified the activation of these pathways by analyzing protein expression levels in mouse liver. The experimental results are as follows: Figure 12 As shown. Western blot experiments showed ( Figure 12 A) Compared with the normal control group, the expression levels of p-AMPK and p-ACC proteins in the HFD-Vehicle group were significantly decreased (P < 0.001), while the expression level of SREBF1 protein was significantly increased (P < 0.05), indicating that the HFD diet can cause lipid metabolism disorders in mice. Compared with the HFD-Vehicle group, the expression levels of p-AMPK and p-ACC proteins in the liver of mice treated with different doses of total saponins of *Abrus precatorius* showed an increasing trend. Among them, the high-dose group (100 mg / kg) significantly increased the phosphorylation levels of AMPK and ACC (P < 0.001, P < 0.01), while the low-dose group (50 mg / kg) of total saponins of *Abrus precatorius* did not show a significant difference in increasing the phosphorylation level of AMPK, but it significantly increased the phosphorylation level of ACC (P < 0.05). The positive control drug pioglitazone did not significantly change the phosphorylation levels of AMPK and ACC. Pioglitazone and high-dose total saponins from *Abrus precatorius* significantly reduced the expression level of SREBF1 protein (P < 0.05). A low-dose total saponins group also showed a decreasing trend, but the difference was not statistically significant. These results indicate that total saponins from *Abrus precatorius* can phosphorylate and activate the AMPK pathway, inhibiting the expression of the downstream target SREBF1 and promoting ACC phosphorylation inactivation, thereby reducing lipid synthesis and alleviating hepatic fat accumulation.

[0091] Regarding the PPAR signaling pathway, we investigated the protein expression levels of PPARα and its downstream target CPT1A. The experimental results are as follows: Figure 12 As shown in Figure B, Western blot analysis revealed that the PPARα protein expression level was significantly lower in the HFD-Vehicle group compared to the normal control group (P < 0.001). Compared to the HFD-Vehicle group, treatment with different doses of total saponins from *Abrus precatorius* showed an increasing trend in the expression levels of PPARα and CPT1A proteins in the mouse liver. The high-dose group of total saponins significantly increased the PPARα protein expression level (P < 0.05), while both the low-dose and high-dose groups significantly increased the CPT1A protein expression level (P < 0.05, P < 0.01). The Western blot results indicate that total saponins from *Abrus precatorius* activate the PPARα pathway, increasing the expression of its downstream target gene CPT1A, thereby improving mitochondrial energy metabolism, accelerating fatty acid oxidation metabolism, and reducing liver fat accumulation.

[0092] 3.9 Summary

[0093] 1. This invention is the first to propose using five main pharmacologically active components as characteristic peaks for total saponins from *Abrus precatorius* and to establish fingerprint spectroscopy to clarify quality standards.

[0094] The extraction process of total saponins from *Abrus precatorius* has been published in the literature. This invention focuses on improving the quality standard of total saponins from *Abrus precatorius*. For the first time, it proposes using five major medicinal components as their characteristic peaks, and setting the peak area of ​​these five characteristic peaks to be greater than 50% of the total peak area, as the chemical fingerprint spectrum and extraction quality standard for total saponins from *Abrus precatorius*. Through further extraction optimization, total saponins from *Abrus precatorius* were successfully extracted into the sample. The results showed that the saponin content in the total saponins from *Abrus precatorius* reached 61.25%, and the positions of the characteristic peaks of the five major medicinal components were clearly identified. The five characteristic peaks were identified as absinin F, daidzein I, dehydrodaidzein I, sophoraemonin III, and absinin SO1.

[0095] 2. This invention focuses on saponin components in the single herb *Abrus precatorius* and provides a novel method for treating metabolic fatty liver disease. It is the first to propose and verify the therapeutic effect of total saponins (ACS) of *Abrus precatorius* on MAFLD in a high-fat diet (HFD) induced animal model, and to conduct pharmacodynamic verification of MAFLD.

[0096] The results showed that in a high-fat diet mouse model, the low and high doses of total saponins from *Abrus precatorius* used in this invention were 50 mg / kg and 100 mg / kg, respectively, which are far lower than the *Abrus precatorius* capsules reported in the literature. [1] The dosages used (780 mg / kg and 2340 mg / kg) achieved the same or even better therapeutic effects. This demonstrates the significant advantages of extracting and enriching the active components of *Abrus precatorius*, which can significantly improve MAFLD at lower doses. It has the advantages of high efficiency, small dosage, and high safety, achieving the purpose of enhancing efficacy and reducing toxicity, and laying the foundation for new drug development.

[0097] In addition, regarding the gut microbiota, total saponins of *Hedychium coronarium* and *Hedychium coronarium* compound capsules also play different roles. *Hedychium coronarium* compound capsules mainly regulate and improve the intestinal microenvironment in the body, by enhancing and restoring the intestinal barrier function and reducing intestinal leakage. Its effect on the gut microbiota is mainly to restore and reduce the abundance of beneficial bacteria such as *Lactobacillus* and *Allobaculum*, while reducing the abundance of harmful bacteria such as *Blautia* and *f. Lachnospiraceae*. Following administration of total saponins from *Abrus precatorius*, numerous changes were observed in the gut microbiota, including the F / B (Firmwallis / Bacteroidetes) ratio, a marker of obesity and metabolic disorders. The proportion of bacteria associated with inflammatory responses (such as *Proteobacteria* and *Escherichia-Shigella*) was significantly reduced, while the abundance of beneficial bacteria associated with short-chain fatty acid production, intestinal barrier function, and lipid metabolism improvement (such as *Lactobacillus*, *Lachnospiraceae_NK4A136_group*, and *Faecalibaculum*) increased. These changes were significantly correlated with hepatic lipid deposition, blood lipid levels, and oxidative stress indicators. This suggests that total saponins from *Abrus precatorius* are not simply "broad-spectrum regulators of bacteria," but rather may selectively regulate the formation of MAFLD-related bacteria through their structural characteristics and metabolic pathways. Therefore, from the perspective of gut microbiota, *Abrus precatorius* compound capsules exhibit a broader regulatory effect on the gut microbiota, far less efficient and precise than the total saponins from *Abrus precatorius* in this invention.

[0098] Other literature [2] Reports indicate that the aqueous extract of *Abrus precatorius* can treat MAFLD. The literature identified 120 compounds in the aqueous extract, highlighting its complex composition and the potential for side effects from the introduction of inactive components, particularly under long-term or high-dose administration. In contrast, the total saponins from *Abrus precatorius* focus on the main active saponin components, selecting five active saponin monomers as characteristic peaks. The area of ​​these five characteristic peaks was greater than 50%, serving as the extraction quality standard. This demonstrates that compared to the aqueous extract, the total saponins from *Abrus precatorius* contain fewer components, have higher purity, stronger activity, and fewer side effects. However, experimental results show that in in vitro and in vivo studies of the aqueous extract, the high-dose group showed even weaker efficacy than the low-dose group. Furthermore, the efficacy was unstable, with significant intra-group variations in TG and TC levels in cells. Some samples showed good efficacy, while others showed almost no efficacy, indicating poor therapeutic stability and potentially low safety with the administration of the aqueous extract.

[0099] The total saponin extract of *Abrus precatorius* of this invention contains clearly defined total saponin components with relatively concentrated target sites, resulting in stable therapeutic efficacy. Furthermore, the therapeutic effect increases in a dose-dependent manner between low and high dose groups. Compared to the aqueous extract, it exhibits higher therapeutic safety, stability, and controllability. This implies a wider therapeutic window, a more defined dose-response relationship, and makes it easier to achieve therapeutic effects superior to *Abrus precatorius* compound capsules and aqueous extract with lower dosages, thus achieving the goal of enhanced efficacy and reduced toxicity, laying the foundation for new drug development.

[0100] References:

[0101] [1] Qi W, Cao X, Chen Y, et al. JiGuCao capsule formula alleviatesmetabolic fatty liver disease by regulating the gut-liver axis and lipidmetabolism. Phytomedicine. 2025;140:156559.

[0102] [2] Liu Chengjun. Preliminary study on the mechanism of action and pharmacodynamic substances of *Abrus precatorius* in the treatment of non-alcoholic fatty liver disease based on chemical composition analysis and metabolomics [D]. Guangxi: Guangxi Normal University, 2024.

[0103] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions without departing from the concept of the present invention are all within the protection scope of the present invention.

Claims

1. Application of total saponin extract of *Abrus precatorius* in the preparation of drugs for the treatment of metabolic-related fatty liver disease.

2. The application according to claim 1, characterized in that, The total saponin extract of *Abrus precatorius* contains daidzein I, sophora japonica saponin III, dehydrodaidzein I, absinthecin F, and absinthecin SO1.

3. The application according to claim 1, characterized in that, The adult dosage of the total saponin extract of *Heliotropium indicum* is 50-300 mg daily.

4. The application according to claim 1, characterized in that, The drug comprises total saponin extract of *Abrus precatorius* and pharmaceutically acceptable carriers or excipients.

5. The application according to claim 1, characterized in that, The dosage form of the drug is oral liquid, pill, granule, capsule, tablet, drop pill, injection or powder.

6. The application according to claim 1, characterized in that, The total saponin extract of *Abrus precatorius* is selected from petroleum ether extract, ethyl acetate extract, n-butanol extract and / or water extract of total saponins of *Abrus precatorius*.

7. The application according to claim 1, characterized in that, The total saponin extract of *Abrus precatorius* can regulate gene expression related to fat synthesis and metabolism, fatty acid oxidation metabolism, and lipid transport and binding.

8. The application according to claim 1, characterized in that, The total saponin extract of *Heliotropium indicum* can improve liver fibrosis levels.

9. The application according to claim 1, characterized in that, The total saponin extract of *Heliotropium indicum* can improve liver inflammation levels.

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