A nutritional composition for lowering lipid and blood sugar and use thereof

Abstract

The present application relates to the technical field of weight loss products, and particularly relates to a nutritional composition for reducing fat and blood sugar and application thereof; the nutritional composition comprises epigallocatechin gallate, animal bifidobacterium lactis BPL1 and phyllanthus emblica extract; the mass concentration ratio of the epigallocatechin gallate, the animal bifidobacterium lactis BPL1 and the phyllanthus emblica extract is 1:0.12-485.20:5.45-87.34; the present application realizes the following breakthroughs by compounding epigallocatechin gallate (EGCG), animal bifidobacterium lactis BPL1 (BPL1) and phyllanthus emblica extract (PEL): multi-target point synergy: simultaneously regulating fat accumulation, lipid droplet dynamics and fatty acid composition, covering multiple pathological links of obesity; reducing dose-dependent risk: reducing the high concentration requirement of single component by compounding, and improving safety.

Description

[0001] This invention claims priority to Chinese Patent Application No. 202510405460.0, filed on April 2, 2025, entitled "A Nutritional Composition for Lowering Lipids and Blood Sugar and Its Application Thereof," the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of weight loss products, and in particular to a nutritional composition for lowering blood lipids and blood sugar and its application. Background Technology

[0003] Obesity refers to a significant degree of overweight and excessive fat accumulation, a condition caused by excessive buildup of body fat, especially triglycerides, resulting from a long-term imbalance between energy intake and energy expenditure. Most available treatments for obesity involve drug interventions that inhibit fat absorption or reduce appetite, thereby affecting food intake. Like other synthetic drugs, these medications (such as orlistat and statins) can produce a variety of side effects.

[0004] The core pathways of lipid metabolism in *C. elegans* (such as the insulin / IGF-1 signaling pathway, AMPK pathway, and mTOR pathway) and core pathways of carbohydrate metabolism (such as the hexokinase pathway, pentose phosphate pathway, and glycolysis-tricarboxylic acid cycle pathway) are highly conserved with those in mammals. The nematodes are transparent, allowing direct observation of the dynamic accumulation of intestinal and subcutaneous fat using fluorescent dyes (such as Nile Red and Oil Red O) or transgenic fluorescent markers (such as the lipid droplet-labeled protein DHS-3::GFP). Specific fluorescent probes (such as 2-NBDG, a fluorescently labeled glucose analog) or transgenic fluorescent markers (such as hexokinase HK-1::GFP fusion protein) can be used to monitor intracellular glucose uptake and changes in the activity of key carbohydrate metabolism enzymes. *C. elegans* has many mutant genes related to lipid and carbohydrate metabolism (such as the daf-2 mutant with increased fat accumulation and the age-1 mutant with significantly altered carbohydrate metabolism rates), which can be used to validate the targets of compounds. With its conserved lipid and glucose regulatory pathways, intuitive visualization of fats and carbohydrates, and efficient genetic manipulation platform, *C. elegans* has become an ideal model for evaluating lipid-lowering and glucose-lowering compounds.

[0005] Epigallocatechin gallate (EGCG) is the most potent catechin, and numerous in vitro and in vivo studies have demonstrated its broad range of biological activities, including lipid-lowering, antioxidant, anti-inflammatory, and neuroprotective effects. However, research indicates that while EGCG exhibits extremely high physiological activity when used alone, it also has limitations such as dose-dependent toxicity, single target, and limited long-term efficacy.

[0006] Bifidobacterium animalis subsp. lactis BPL1 (BPL1) is a probiotic strain isolated from the intestines of healthy humans. Studies have shown that BPL1 can regulate lipid metabolism and reduce fat accumulation. Phyllanthus emblica is a traditional Tibetan medicine, the dried, ripe fruit of the Euphorbiaceae plant Phyllanthus emblica. Numerous studies have shown that Phyllanthus emblica extract (PEL) has antioxidant, anti-inflammatory, lipid-lowering, insulin-sensitivity-improving, and immunity-enhancing effects.

[0007] With the continuous rise in the incidence of obesity, the market demand for weight loss-related products continues to grow. Currently, the market is flooded with various lipid-lowering and blood sugar-lowering products, but there are no reports of combining EGCG, PEL, and BPL1. Summary of the Invention

[0008] In view of the shortcomings of the prior art, one of the objectives of the present invention is to provide a nutritional composition for lowering blood lipids and blood sugar; another objective of the present invention is to provide the use of the composition in the preparation of medicines for the prevention or treatment of hyperlipidemia, hyperglycemia, and obesity.

[0009] One of the objectives of this invention is achieved through the following technical solution: A nutritional composition for lowering blood lipids and blood sugar includes epigallocatechin gallate, Bifidobacterium animalis subsp. lactis BPL1, and Phyllanthus emblica extract; wherein the molar concentration of epigallocatechin gallate is 50-200 μM / L (mass concentration is 0.0229-0.0917 mg / mL), and the cell density of Bifidobacterium animalis subsp. lactis BPL1 in the well plate is 10-1. 6 -10 9 Cells / plate (mass concentration of 0.0111-11.1111 mg / mL) and Phyllanthus emblica extract (mass concentration of 0.5-2 mg / mL) are used. That is, the mass concentration ratio of epigallocatechin gallate, Bifidobacterium animalis subsp. lactis BPL1, and Phyllanthus emblica extract is 1:0.12-485.20:5.45-87.34.

[0010] In a preferred embodiment, the mass concentration ratio of epigallocatechin gallate, Bifidobacterium animalis subsp. lactis BPL1, and Phyllanthus emblica extract is 1:1.6:23.3.

[0011] In a preferred embodiment, the mass concentrations of epigallocatechin gallate, Bifidobacterium animalis subsp. lactis BPL1, and Phyllanthus emblica extract are 0.0458 mg / mL, 0.0746 mg / mL, and 1.0659 mg / mL, respectively.

[0012] Furthermore, the Bifidobacterium lactis subsp. BPL1 comprises dead bacteria, live bacteria, or a mixture of both.

[0013] Furthermore, the purity of the epigallocatechin gallate is 92-96%, and the content of β-glucosidogalactan in the amla extract is 8-12% w / w.

[0014] The second objective of this invention is achieved through the following technical solution: The above composition is used to prepare a medicine for the prevention or treatment of hyperlipidemia, hyperglycemia, or obesity.

[0015] Furthermore, the nutritional composition is used in the preparation of drugs that reduce triglyceride, cholesterol ester, glucose, and glycogen levels; reduce the number of lipid droplets and the overall diameter of lipid droplets; promote triglyceride lipase-1 expression; and inhibit fatty acid synthase-1 expression. Experiments have demonstrated that this nutritional composition produced the following therapeutic effects in a high-glucose-induced obese Caenorhabditis elegans model: Body bending frequency, head swing frequency, and average movement rate of obese *C. elegans*; triglyceride, cholesterol ester, glucose, and glycogen levels in obese *C. elegans*; body size, number of lipid droplets, and overall diameter of lipid droplets in obese *C. elegans*; expression levels of triglyceride lipase-1 and fatty acid synthase-1 in obese *C. elegans*.

[0016] Furthermore, if the Bifidobacterium lactis subsp. BPL1 in the drug is a live bacterium, then the content of the live bacterium in the drug is 10. 9 CFU / g-10 11 CFU / g; If the Bifidobacterium lactis subsp. 1, a dead bacterium, is used in the drug, then the content of the dead bacterium in the drug is 10. 9 cells / g-10 11 cells / g.

[0017] The beneficial effects of this invention are as follows: Using a high-glucose-induced obese *C. elegans* model, intervention with a nutritional composition containing EGCG, BPL1, and PEL significantly inhibited fat accumulation, reduced glycogen content, and increased the activity of fat-degrading enzymes in the obese nematodes. Specifically, this was manifested in a significant increase in head-shaking frequency, swallowing frequency, and average movement speed of the obese nematodes, and a significant reduction in nematode size. This also indicates that the intervention of each test substance had no negative effects on the nematodes. Quantitative analysis of the expression of fat metabolism-related enzymes and the presence of lipid droplets around the intestine in obese nematodes revealed the presence of EGCG, BPL1, and PEL. The nutritional composition of L significantly increased the expression of triglyceride lipase-1 (ATGL-1), inhibited the expression of fatty acid synthase-1 (FASN-1), and reduced the overall diameter of periintestinal lipid droplets after intervention, indicating that the nutritional composition can reduce fat accumulation in obese nematodes. Biochemical results showed that the nutritional composition could significantly reduce the levels of TG, cholesterol esters, glucose, and glycogen in obese nematodes. Therefore, the nutritional composition containing EGCG, BPL1, and PEL may play a role in lowering lipids and blood sugar by inhibiting the activity of fat synthesis-related enzymes and increasing the rate of fat metabolism.

[0018] This invention achieves the following breakthroughs by combining epigallocatechin gallate (EGCG), Bifidobacterium animalis subsp. lactis 1 (BPL1), and Phyllanthus emblica extract (PEL): Multi-target synergistic effect: Simultaneously regulates fat accumulation (reducing glucose and glycogen levels, activating triglyceride lipase-1, and downregulating fatty acid synthase-1), lipid droplet dynamics (inhibiting fusion), and fatty acid composition (increasing the ratio of unsaturated fatty acids), covering multiple pathological aspects of obesity. Reduced dose-dependent risk: The combination reduces the need for high concentrations of single components (such as EGCG), improving safety. Attached Figure Description

[0019] Figure 1 The effects of treatment with EGCG, BPL1, PEL and their combinations on lipid content in nematodes; Figure 2 The effect of treatment with EGCG, BPL1, PEL and their combinations on the body bending frequency of nematodes; Figure 3 The effects of treatment with EGCG, BPL1, PEL and their combinations on the head-shaking frequency of nematodes; Figure 4 The effect of treatment with EGCG, BPL1, PEL and their combinations on the average motility rate of nematodes; Figure 5 The effects of treatment with EGCG, BPL1, PEL and their combinations on the body size of nematodes; Figure 6 The effects of treatment with EGCG, BPL1, PEL and their combinations on triglyceride levels in nematodes; Figure 7 Effects of treatment with EGCG, BPL1, PEL and their combinations on cholesterol ester levels in nematodes Figure 8 The effects of treatment with EGCG, BPL1, PEL and their combinations on glucose levels in nematodes; Figure 9 The effects of treatment with EGCG, BPL1, PEL and their combinations on glycogen levels in nematodes; Figure 10 The effects of treatment with EGCG, BPL1, PEL and their combinations on the expression of triglyceride lipase in nematodes; Figure 11 The effects of treatment with EGCG, BPL1, PEL and their combinations on fatty acid synthase expression in nematodes; Figure 12 The effect of treatment with EGCG, BPL1, PEL and their combinations on the number of lipid droplets in nematodes; Figure 13 The effect of treatment with EGCG, BPL1, PEL and their combinations on the average size of lipid droplets in nematodes; Figure 14 The effects of treatment with EGCG, BPL1, PEL and their combinations on the lipid droplet size distribution of nematodes; Figure 15 The effects of treatment with EGCG, BPL1, PEL and their combinations on the fatty acid composition of nematodes; Figure 16 The effect of treatment with EGCG, BPL1, PEL and their combinations on the ratio of unsaturated fatty acids in nematodes; Figure 17 The synergistic effect of the composition of the present invention on glucose levels in obese nematodes; Figure 18 The composition of this invention is used to treat the synergistic effect of ATGL-1 expression in obese nematodes. Detailed Implementation

[0020] The following is a further explanation with reference to specific implementation methods: Ingredients: The epigallocatechin gallate ester in the examples has a purity of 94%, and the β-glucosidogalactan content in the amla extract is 10% w / w.

[0021] First, culture blank control group, model group, EGCG group (divided into 50, 100, and 200 μM / L groups), and BPL1 group (divided into 3×10 groups). 7 3×10 8 3×10 9 0.3358×10 7 0.6716×107 and 1.3432×10 7 The groups included cells / plate, PEL (divided into 0.5330, 1.0659, and 2.1319 mg / mL groups), and a combination group containing N2 nematodes.

[0022] 1) Blank control group N2 nematode eggs were cultured on blank control medium at 20℃ for 3 days. The blank control (OP50) medium was prepared by directly spreading E. coli OP50 bacterial suspension onto NGM, drying it, sealing it, and storing it at 4℃ for later use.

[0023] 2) Model Group N2 nematode eggs were cultured on the model group (high-glucose modeling) medium at 20℃ for 3 days. The high-glucose modeling medium was prepared as follows: 400 mL of NGM medium was prepared, sterilized, and 8 mL of 2.5 mol / L glucose was added. After mixing and pouring, E. coli OP50 bacterial suspension was directly spread onto the NGM medium. After drying, the medium was sealed and stored at 4℃ for later use.

[0024] 3) EGCG group N2 nematode eggs were cultured on EGCG sample treatment medium at 20℃ for 3 days. The EGCG sample treatment medium was prepared by mixing E. coli OP50 bacterial suspensions containing EGCG samples at molar concentrations of 50, 100, and 200 μM / L. The bacterial suspensions were then spread onto the NGM model, dried, sealed, and stored at 4℃ for later use.

[0025] 4) BPL1 group N2 nematode eggs were cultured on BPL1 sample treatment medium at 20℃ for 3 days. The BPL1 sample treatment medium was prepared by adjusting the cell density in the wells to 10-1. 7 10 8 10 9 Prepare E. coli OP50 bacterial suspension containing BPL1 sample using cells / plate, spread the bacterial suspension on the NGM model, air dry, seal with a membrane, and store at 4℃ for later use.

[0026] 5) PEL Group N2 nematode eggs were cultured on PEL sample treatment medium at 20℃ for 3 days. The PEL sample treatment medium was prepared as follows: E. coli OP50 bacterial suspensions containing PEL samples were prepared at concentrations of 0.5330, 1.0659, and 2.1319 mg / mL. The bacterial suspensions were spread onto the NGM model, dried, sealed, and stored at 4℃ for later use.

[0027] 6) Composition group N2 nematode eggs were cultured on the combined treatment medium at 20℃ for 3 days. The combined treatment medium was prepared as follows: E. coli OP50 bacterial suspension was prepared with EGCG concentration of 0.0458 mg / mL, BPL1 concentration of 0.0746 mg / mL, and PEL concentration of 1.0659 mg / mL. The bacterial suspension was spread onto the NGM model, dried, sealed, and stored at 4℃ for later use.

[0028] II. Effects of Nile Red Staining on Lipid Content in N2 Nematodes: Calculation of the Effects of Different Concentrations of EGCG, BPL1, and PEL on Sample Treatment. Experimental methods: N2 nematodes from the blank control group, model group, EGCG group, BPL1 group, and PEL group were washed until the supernatant was clear. 50 μL of anesthetic was added to the remaining 1 mL, the supernatant was discarded, and 1 mL of 40% isopropanol was added. After 3 min, the supernatant was discarded, and 1 mL of 3 μg / mL Nile Red working solution was added in the dark. The mixture was mixed and incubated at room temperature for 2 h. The staining solution was washed off, slides were prepared, and photographs were taken. ImageJ software was used for quantitative analysis of the images.

[0029] As shown in Table 1, the Nile Red fluorescence intensity in the model group was significantly increased, indicating that high-glucose modeling led to increased fat accumulation in nematodes. After EGCG intervention, the fat content in nematodes decreased to the level of the blank control group, with 100 and 200 μM / L showing the best effects and no significant difference between the two. Therefore, 100 μM / L (0.0458 mg / mL) was used in subsequent experiments.

[0030] Table 1. Effects of different concentrations of EGCG on lipid content in nematodes.

[0031] As shown in Table 2, the Nile Red fluorescence intensity in the model group increased significantly, indicating that high sugar modeling led to increased lipid accumulation in nematodes. After 3×10 7 3×10 8 After BPL1 intervention in cells / plates, the fluorescence intensity decreased significantly and the effect was similar, indicating that BPL1 could significantly reduce lipid accumulation in nematodes, restoring it to the control group level. For safety reasons, the minimum effective concentration was subsequently set at 3 × 10⁻⁶. 7 cells / plate.

[0032] Table 2. Effects of different concentrations of BPL1 on lipid content in nematodes.

[0033] Although the initial screening showed 3×10 7Cells / plate BPL1 has shown significant lipid-lowering effects, but considering the principle of "lowest effective concentration" (i.e., selecting the smallest dose that achieves a therapeutic effect), secondary validation was conducted at a finer concentration gradient. As shown in Table 3, 0.3358 × 10⁻⁶ BPL1 was used. 7 0.6716×10 7 and 1.3432×10 7 The three concentrations of cells / plate were used for secondary screening of BPL1, and the results showed that 0.6716 × 10⁻⁶ was the optimal concentration. 7 BPL1 cells / plate showed the best lipid-lowering effect, so this was selected as the lowest effective concentration, namely 0.0746 mg / mL.

[0034] Table 3. Effects of different concentrations of BPL1 on lipid content in nematodes.

[0035] As shown in Table 4, the Nile Red fluorescence intensity in the model group increased significantly, indicating that high-glucose modeling led to increased lipid accumulation in nematodes. Treatment with 0.5330, 1.0659, and 2.1319 mg / mL PEL significantly reduced fluorescence intensity, indicating that PEL significantly reduced lipid accumulation in nematodes. Treatment with 1.0659 and 2.1319 mg / mL PEL restored lipid accumulation to the control group level. Therefore, the lowest effective concentration was selected as 1.0659 mg / mL, which is consistent with the reported result of 1 mg / mL in the literature.

[0036] Table 4. Effects of different concentrations of PEL on lipid content in nematodes.

[0037] Third, the optimal concentration for Nile Red staining was determined (i.e., EGCG 100 μM / L and BPL1 0.6716 × 10⁻⁶) as screened above. 7 Effects of EGCG, BPL1, PEL and their combinations on lipid content in N2 nematodes (cells / plate and PEL 1.0659 mg / mL; the same below) Experimental methods: N2 nematodes were cultured according to (I), including a blank control group, a model group, an EGCG group (100 μM / L), and a BPL1 group (0.6716 × 10⁻⁶). 7 The groups were classified into three groups: cells / plate, PEL group (1.0659 mg / mL), and combination group (EGCG 100 μM / L, BPL 10.6716 × 10⁻⁶). 7Cells / plate and PEL 1.0659 mg / mL N2 nematodes were used to wash the nematodes until the supernatant was clear. 50 μL of anesthetic was added to the remaining 1 mL, the supernatant was discarded, and 1 mL of 40% isopropanol was added. After 3 min, the supernatant was discarded again. In the dark, 1 mL of 3 μg / mL Nile Red working solution was added, mixed well, and incubated at room temperature for 2 h. The staining solution was washed off, slides were prepared, and photographs were taken. Quantitative analysis of the images was performed using ImageJ software.

[0038] like Figure 1 As shown, the Nile Red fluorescence intensity in the model group increased significantly, indicating that high-sugar modeling led to increased lipid accumulation in nematodes. After treatment with EGCG, BPL1, PEL, or a combination thereof, the Nile Red fluorescence intensity decreased significantly, indicating that the total lipid accumulation in the nematodes was improved after sample treatment.

[0039] IV. Effects of optimal concentrations of EGCG, BPL1, PEL, and their combinations on body bending frequency, head swing frequency, average movement speed, and body size of *Nematodea obeseis*. Experimental methods: N2 nematodes were cultured according to (I), including a blank control group, a model group, an EGCG group (100 μM / L), and a BPL1 group (0.6716 × 10⁻⁶). 7 The groups were classified into three groups: cells / plate, PEL group (1.0659 mg / mL), and combination group (EGCG 100 μM / L, BPL 10.6716 × 10⁻⁶). 7 Cells / plate and PEL 1.0659 mg / mL) N2 nematodes.

[0040] Body bending measurement: Under a biological microscope, the number of times a nematode completes a sinusoidal movement relative to its long axis is recorded as one complete bending motion. The number of body bending motions of the nematode within 60 seconds is recorded.

[0041] Head swing measurement: The number of times the nematode's head swings within 30 seconds is observed and recorded under a biological microscope. The standard for measurement is that the nematode's head swings from the left to the right and then back to the left, which is recorded as one swing.

[0042] Movement speed measurement: 20-30 NL5901 nematodes of the same size were selected from each group and placed in an NGM containing the test substance for video recording. The video was recorded using the Wormlab nematode video acquisition system, and the average movement speed of the nematodes was analyzed.

[0043] Size determination: N2 nematodes from each group were washed with M9 solution in centrifuge tubes until sterile supernatant remained. The nematodes were anesthetized and photographed under a microscope. At least 20 nematodes were observed in each group, and each experiment was repeated at least three times. ImageJ software was used for quantitative analysis of nematode area.

[0044] like Figure 2 , 3 4. The overall motility of the model group nematodes was impaired, manifested by decreased body bending frequency, decreased head swing frequency, and slowed average movement speed. However, after intervention with EGCG, BPL1, PEL, or a combination thereof, the above indicators of obese nematodes at least recovered to the motility level of normal nematodes. Among them, the combination group showed a significantly increased head swing frequency in obese nematodes compared to the other groups, indicating that the combination of EGCG, BPL1, and PEL may have a synergistic effect in increasing the head swing frequency of obese nematodes.

[0045] like Figure 5 As shown, the body area of ​​nematodes in the model group increased significantly. After intervention with EGCG, BPL1, PEL, or a combination of these agents, the body size development of nematodes was effectively controlled. The effects were more significant in the EGCG group and the combination of these agents, and there was no difference in body area between the two groups, suggesting that EGCG may be an important factor in regulating the obesity-related body size of nematodes.

[0046] Therefore, EGCG, BPL1, PEL, and their combination may reduce fat accumulation and body surface area in obese nematodes by improving their motility, increasing heat production and energy metabolism.

[0047] 5. Effects of optimal concentrations of EGCG, BPL1, PEL, and their combinations on TG, cholesterol ester, glucose, and glycogen levels in obese nematodes. Refer to (I) to culture N2 nematodes, including blank group, model group, EGCG group (100 μM / L), and BPL1 group (0.6716 × 10⁻⁶). 7 Cells / plate), PEL group (1.0659 mg / mL), and combination group (EGCG 100 μM / L, BPL1 0.6716 × 10⁻⁶) 7 Cells / plate and PEL 1.0659 mg / mL) N2 nematodes.

[0048] TG, cholesterol ester, and glucose levels were determined as follows: N2 nematodes from each group were homogenized using an automated tissue homogenizer, centrifuged at low temperature (4℃, 8000 r / min, 10 min), and the supernatant was collected and stored at 4℃ for analysis. TG and glucose levels were determined according to the instructions provided with the kits (purchased from Nanjing Jiancheng Biotechnology Institute) and the cholesterol ester kit (Beyotime). Results were standardized by protein concentration.

[0049] Glycogen level determination: N2 nematodes from each group were washed in centrifuge tubes with M9 solution until sterile supernatant remained, with a final 0.5 mL supernatant retained. 0.5 mL of 0.1% Lugol's iodine solution was added, and the tubes were allowed to stand for 2 minutes to anesthetize them before photographing. At least 20 nematodes were observed in each group, and each experiment was repeated at least three times. ImageJ software was used for quantitative analysis of the images.

[0050] like Figure 6 As shown, compared with the blank control group, the TG level in the model group of nematodes was significantly increased. After intervention with EGCG, BPL1, PEL, or a combination of these treatments, the TG level in obese nematodes was significantly reduced, approaching the level of the control group (mean 0.53 mmol / g prot). The EGCG group showed the best effect (mean 0.48 mmol / g prot), followed by the combination group and the PEL group. The decrease in TG level may be one of the main reasons for the decrease in total lipid content in obese nematodes, with EGCG and PEL being the main contributors to the effect of the combination treatments.

[0051] like Figure 7 As shown, compared with the blank control group, the cholesterol ester level in the model group nematodes was significantly increased, by approximately 1.4 g / g prot, indicating successful model establishment. After intervention with EGCG, BPL1, PEL, or a combination thereof, the cholesterol ester content in obese nematodes was significantly reduced. The BPL1 group (mean 0.78 g / g prot) and the PEL group (mean 0.76 g / g prot) showed better effects than the control group (mean 1.1 g / g prot). The reduction in cholesterol ester levels may also be one of the main reasons for the decrease in total lipid content in obese nematodes, with the lipid-lowering effect ranking as follows: PEL, BPL1 > combination > EGCG.

[0052] like Figure 8 , 9 As shown, compared with the blank control group, the accumulation of glucose and glycogen in the model group of nematodes was significantly increased. Overall, the glucose and glycogen content in the nematodes decreased to varying degrees after intervention with EGCG, BPL1, PEL, or the combination. The EGCG group and the combination group significantly reduced glucose and glycogen levels, while BPL1 and PEL had no significant effect. The results indicate that the combination promotes glucose metabolism in obese nematodes, with EGCG likely being the main contributor.

[0053] In summary, EGCG, BPL1, PEL, and their combinations have the effect of reducing glycolipid levels in obese nematodes. Compared with the individual components, the combinations have significant effects on TG, cholesterol esters, glucose, and glycogen levels.

[0054] 6. Effects of optimal concentrations of EGCG, BPL1, PEL, and their combinations on the expression of ATGL-1 and FASN-1 and the presence of perigut lipid droplets in *C. obese*. ATGL-1 expression determination: Following procedure (I), VA20 mutant nematodes were cultured, including a blank control group, a model group, an EGCG group (100 μM / L), and a BPL1 group (0.6716 × 10⁻⁶). 7 Cells / plate), PEL group (1.0659 mg / mL), and combination group (EGCG 100 μM / L, BPL1 0.6716 × 10⁻⁶) 7 Cells / plate and PEL 1.0659 mg / mL) VA20 mutant nematodes were fed until day 3. Uniformly sized nematodes were picked and placed on a glass slide, anesthetic was added, and after the nematodes became rigid, a coverslip was placed on top, and the slide was photographed at 40× under a fluorescence microscope. At least 15 nematodes were used in each group, and the experiment was repeated three times. Quantitative fluorescence analysis was then performed using ImageJ software.

[0055] FASN-1 expression determination: Refer to (I) for the culture of pfasn-1::GFP transgenic nematode AG400, including blank control group, model group, EGCG group (100 μM / L), and BPL1 group (0.6716 × 10⁻⁶). 7 Cells / plate), PEL group (1.0659 mg / mL), and combination group (EGCG 100 μM / L, BPL1 0.6716 × 10⁻⁶) 7 Cells / plate and PEL 1.0659 mg / mL) pfasn-1::GFP transgenic nematodes AG400; after feeding for 3 days, uniformly sized nematodes were picked and placed on a glass slide, anesthetic was added, and after the nematodes became rigid, a coverslip was placed on top, and the slide was placed under a fluorescence microscope for 20× and 40× imaging. At least 15 nematodes were used in each group, and the experiment was repeated three times. Quantitative fluorescence analysis was then performed using ImageJ software.

[0056] Periintestinal lipid droplet assay: Refer to (I) for culturing ZXW618 mutant nematodes, including a blank control group, a model group, an EGCG group (100 μM / L), and a BPL1 group (0.6716 × 10⁻⁶). 7 Cells / plate), PEL group (1.0659 mg / mL), and combination group (EGCG 100 μM / L, BPL1 0.6716 × 10⁻⁶) 7Cells / plate and PEL 1.0659 mg / mL) ZXW618 mutant nematodes were fed until day 3. Uniformly sized nematodes were picked and placed on a glass slide, anesthetic was added, and after the nematodes became rigid, a coverslip was placed on top. A laser confocal microscope was used with a 488 nm excitation filter and a 500 nm emission filter, and photographs were taken at 40× magnification. Each group contained at least 15 nematodes, and the experiment was repeated three times. Quantitative fluorescence analysis was then performed using ImageJ software.

[0057] like Figure 10 As shown, changes in ATGL-1 expression levels are represented by quantitative fluorescence results. Stronger fluorescence intensity indicates higher ATGL-1 activity, signifying accelerated lipolysis and a beneficial effect on high-sugar diet-induced intestinal steatosis and lipid homeostasis. Compared to the blank control group (fluorescence intensity 21), the model group (fluorescence intensity 12) showed significantly reduced ATGL-1 expression. After intervention with EGCG, BPL1, PEL, and a combination of these treatments, ATGL-1 activity was greatly improved and restored, with the combination group showing the best effect, recovering to normal levels (fluorescence intensity 21). Figure 11 As shown, changes in FASN-1 expression levels are represented by quantitative fluorescence results. Stronger fluorescence intensity indicates higher FASN-1 activity, signifying accelerated lipid synthesis. Compared to the blank control group (fluorescence intensity 11), the model group (fluorescence intensity 15) showed significantly increased FASN-1 expression levels. Intervention with EGCG, BPL1, PEL, and their combinations significantly reduced FASN-1 expression levels, with the combination group showing the best effect.

[0058] In summary, the combination of EGCG, BPL1, and PEL suggests that the components may have a synergistic effect in regulating the expression of ATGL-1 and FASN-1, effectively affecting the breakdown and synthesis of fat, thereby influencing lipid accumulation.

[0059] like Figure 12 As shown, compared to the model group (mean number of lipid droplets 55), intervention with BPL1, PEL, and their combinations significantly reduced the number of lipid droplets in obese nematodes (mean numbers of lipid droplets were 46, 41, 42, and 39, respectively). Figure 13 As shown, compared to the model group (average lipid droplet diameter 4.14 μm), intervention with EGCG, BPL1, PEL, and their combinations significantly reduced the size of lipid droplets in *C. obeseis* (average droplet diameters were 3.47, 3.46, 3.32, and 3.51 μm, respectively). Figure 14As shown, compared to the model group, intervention with EGCG, BPL1, PEL, and their combinations significantly reduced the proportion of large-diameter lipid droplets and the number of large-diameter lipid droplets in obese nematodes. Previous studies have shown that a larger specific surface area of ​​lipid particles is more conducive to fat consumption. Therefore, it is speculated that the lipid-lowering effect of EGCG, BPL1, PEL, and their combinations may be related to increasing the proportion of small-diameter lipid particles and increasing the specific surface area of ​​lipid particles, thereby promoting fat consumption.

[0060] 7. Effects of optimal concentrations of EGCG, BPL1, PEL, and their combinations on the fatty acid composition of *C. obese*. Refer to (I) for culturing N2 nematodes, including a blank control group, a model group, an EGCG group (100 μM / L), and a BPL1 group (0.6716 × 10⁻⁶). 7 The groups were classified into three groups: cells / plate, PEL group (1.0659 mg / mL), and combination group (EGCG 100 μM / L, BPL 10.6716 × 10⁻⁶). 7 Cells / plate and PEL 1.0659 mg / mL N2 nematodes were washed thoroughly with M9 buffer and placed in 2 mL EP tubes. Then, GC-MS analysis was performed according to the method described in the literature.

[0061] like Figure 15 As shown, compared with the blank control group OP50, the levels of C14:0, C15:0, C16:0, C17:1, C18:3, C20:0, C20:3, and C22:1 in the model group were significantly increased, while the levels of C18:1, C20:1, and C20:2 were significantly decreased. Intervention with EGCG, BPL1, PEL, and their combination could reverse the changes in the content of various fatty acids caused by a high-sugar diet and maintain the stability of the fatty acid composition of each carbon number. Among them, the combination had a significant effect on restoring the normal levels of all fatty acids.

[0062] like Figure 16 As shown, compared with the blank control group, the unsaturated fatty acid ratios (C16:1 / C16:0, C18:1 / C18:0, and C20:1 / C20:0) of nematodes in the model group were significantly reduced. Intervention with EGCG, BPL1, PEL, and their combinations increased these unsaturated fatty acid ratios. The combination group and the BPL1 group showed better results, suggesting that BPL1 plays a major role in regulating the fatty acid composition of nematodes through these combinations.

[0063] 8. The synergistic effect of the composition of the present invention on glucose levels in obese nematodes. N2 nematodes were cultured in blank control group, model group, EGCG group, BPL1 group, PEL group and combination group.

[0064] 1) Blank control group N2 nematode eggs were cultured on blank control medium at 20℃ for 3 days. The blank control (OP50) medium was prepared by directly spreading E. coli OP50 bacterial suspension onto NGM, drying it, sealing it, and storing it at 4℃ for later use.

[0065] 2) Model Group N2 nematode eggs were cultured on the model group (high-glucose modeling) medium at 20℃ for 3 days. The high-glucose modeling medium was prepared as follows: 400 mL of NGM medium was prepared, sterilized, and 8 mL of 2.5 mol / L glucose was added. After mixing and pouring, E. coli OP50 bacterial suspension was directly spread onto the NGM medium. After drying, the medium was sealed and stored at 4℃ for later use.

[0066] 3) EGCG group N2 nematode eggs were cultured on EGCG sample treatment medium at 20℃ for 3 days. The EGCG sample treatment medium was prepared as follows: E. coli OP50 bacterial suspension containing EGCG sample was prepared at a concentration of 1.1863 mg / mL. The bacterial suspension was spread onto the NGM model, dried, sealed, and stored at 4℃ for later use.

[0067] 4) BPL1 Group N2 nematode eggs were cultured on BPL1 sample treatment medium at 20℃ for 3 days. The BPL1 sample treatment medium was prepared as follows: E. coli OP50 bacterial suspension containing BPL1 sample was prepared at a concentration of 1.1863 mg / mL. The bacterial suspension was spread onto the NGM model, dried, sealed, and stored at 4℃ for later use.

[0068] 5) PEL Group N2 nematode eggs were cultured on PEL sample treatment medium at 20℃ for 3 days. The PEL sample treatment medium was prepared as follows: E. coli OP50 bacterial suspension containing PEL samples was prepared at a concentration of 1.1863 mg / mL. The bacterial suspension was spread onto the NGM model, dried, sealed, and stored at 4℃ for later use.

[0069] 6) Composition group N2 nematode eggs were cultured on the combined treatment medium at 20℃ for 3 days. The combined treatment medium was prepared as follows: E. coli OP50 bacterial suspension was prepared with EGCG concentration of 0.0458 mg / mL, BPL1 concentration of 0.0746 mg / mL, and PEL concentration of 1.0659 mg / mL. The bacterial suspension was spread onto the NGM model, dried, sealed, and stored at 4℃ for later use.

[0070] Glucose level determination: N2 nematodes from each group were homogenized using an automated tissue homogenizer, centrifuged at low temperature (4℃, 8000 r / min, 10 min), and the supernatant was collected and stored at 4℃ for analysis. Glucose levels were determined according to the instructions provided with the glucose kit (purchased from Nanjing Jiancheng Biotechnology Institute), and the results were standardized by protein concentration.

[0071] The measurement results are as follows Figure 17 As shown, the glucose levels in the EGCC group, BPL1 group, and PEL group increased by 19.4%, 25.5%, and 20.4% respectively compared to the model group, with a total increase of 65.3%. The composition of this invention increased the glucose level in the model group by 70.8%, demonstrating that the composition of this invention, by combining EGCC, BPL1, and PEL, has a synergistic effect.

[0072] 9. The synergistic effect of the composition of the present invention on ATGL-1 expression in *C. obese*. ATGL-1 expression determination: Following the guidelines in section VIII, "Synergistic effect of the composition of this invention on glucose levels in obese nematodes," blank control group, model group, EGCG group (300 μM / L), and BPL1 group (2.0148 × 10⁻⁶) were cultured. 7 Cells / plate), PEL group (3.1977 mg / mL), and combination group (EGCG 100 μM / L, BPL1 0.6716 × 10⁻⁶) 7 Cells / plate and PEL 1.0659 mg / mL) VA20 mutant nematodes were fed until day 3. Uniformly sized nematodes were picked and placed on a glass slide, anesthetic was added, and after the nematodes became rigid, a coverslip was placed on top, and the slide was photographed at 40× under a fluorescence microscope. At least 15 nematodes were used in each group, and the experiment was repeated three times. Quantitative fluorescence analysis was then performed using ImageJ software.

[0073] The measurement results are as follows Figure 18 As shown, the glucose levels in the EGCC group, BPL1 group, and PEL group increased by 19.5%, 28.2%, and 27.5% respectively compared to the model group, with a total increase of 75.2%. Furthermore, the composition of this invention increased the glucose level in the model group by 76.0%, further demonstrating that the composition of this invention, by combining EGCC, BPL1, and PEL, has a synergistic effect.

[0074] 10. Summary This invention innovatively develops a multi-component synergistic nutritional composition that achieves dynamic regulation of lipid and glucose metabolism through a triple synergistic mechanism of "synthesis inhibition-lipolysis promotion-structural remodeling". Compared with single components, this composition exhibits the following breakthrough advantages: 1. Exercise-metabolic synergistic activation mechanism The composition significantly increases the head-swing frequency of obese nematodes, effectively reversing the impairment of exercise capacity caused by glucose toxicity and establishing a positive cycle of "exercise metabolism-lipid consumption," an effect that is significantly better than that of any single component.

[0075] 2. The dynamic balance of "catabolism promotion-synthesis inhibition" regulated by dual pathways of lipid metabolism. The composition significantly upregulated ATGL-1 expression (9% higher than the EGCG group), activating lipolysis; and significantly downregulated FASN-1 levels (15% lower than the EGCG group), inhibiting lipogenesis. The synergistic effect of the two pathways reduced cholesterol ester accumulation to 69% of that in the EGCG group, achieving precise enhancement of metabolic regulation. Simultaneously, after intervention with the composition, the number of lipid droplets in obese nematodes decreased by 16% compared to the EGCG group.

[0076] 3. Dynamically regulate the composition and structure of fatty acid spectra The composition dynamically regulates the fatty acid profile, significantly reducing the C14:0-C20:0 content (6-28% lower than the EGCG group), effectively reducing the level of saturated fatty acids; the C20:1 / C20:0 ratio is increased to 1.37 times that of the EGCG group, promoting the conversion of unsaturated fatty acids and helping to reduce the accumulation of bad fats.

[0077] 4. Component functional complementarity design BPL1 and PEL exhibited synergistic advantages in specific indicators: for example, BPL1 showed the best effect in improving the efficiency of fatty acid unsaturation conversion (e.g., the C20:1 / C20:0 and C20:2 / C20:0 ratios in the BPL1 group were 24% and 28% higher than those in the EGCG group, respectively); the PEL group showed outstanding effects in regulating lipid droplet microstructure (the number of lipid droplets was reduced by 10% compared to the EGCG group). The composition also showed significant effects on all of the above indicators, indicating that the composition forms a regulatory network covering the entire lipid metabolism cycle through a three-tiered effect of "synthesis inhibition - lipolysis promotion - structural remodeling".

[0078] Compared to individual components, this composition utilizes a triple synergistic mechanism of exercise-induced metabolic activation, dual-pathway enzyme regulation, and lipidome remodeling to comprehensively target all aspects of glucose and lipid metabolism. It is particularly suitable for developing drugs to prevent and treat obesity-related metabolic syndrome, providing an innovative solution to industry challenges such as the limited target of existing formulations.

[0079] The embodiments and descriptions above are merely illustrative of the principles and preferred embodiments of the present invention. Various changes and modifications may be made to the present invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed.

Claims

1. A composition for lowering lipids and blood sugar, characterized in that, It is composed of epigallocatechin gallate, Bifidobacterium animalis subsp. lactis BPL1, and Phyllanthus emblica extract; the mass concentration ratio of epigallocatechin gallate, Bifidobacterium animalis subsp. lactis BPL1, and Phyllanthus emblica extract is 1:1.6:23.3; the purity of epigallocatechin gallate is 92-96%, and the content of β-glucosidogalactan in Phyllanthus emblica extract is 8-12% w / w.

2. The composition according to claim 1, characterized in that: The mass concentrations of epigallocatechin gallate, Bifidobacterium animalis subsp. lactis BPL1, and Phyllanthus emblica extract were 0.0458 mg / mL, 0.0746 mg / mL, and 1.0659 mg / mL, respectively.

3. The composition according to claim 1, characterized in that: The animal Bifidobacterium lactis subspecies BPL1 includes dead bacteria, live bacteria, or a mixture of both.

4. The use of the composition according to any one of claims 1-3 in the preparation of a medicament for treating hyperlipidemia, hyperglycemia or obesity.

5. The application according to claim 4, characterized in that, If the Bifidobacterium lactis subsp. BPL1 in the drug is a live bacterium, then the content of the live bacterium in the drug is 10. 9 CFU / g-10 11 CFU / g; If the Bifidobacterium lactis subsp. 1, a dead bacterium, is used in the drug, then the content of the dead bacterium in the drug is 10. 9 cells / g-10 11 cells / g.

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