A composition containing litsea glutinella extract and a preparation method and application thereof

Abstract

The present application belongs to the technical field of health food, and particularly relates to a composition containing Litsea chenii extract and a preparation method and application thereof. The composition disclosed by the present application takes Litsea chenii extract, American ginseng extract, mulberry leaf extract, radix astragali extract and chromium-rich yeast as main raw materials, optimizes the proportion and dosage of the composition through animal experiments, and proves through animal experiments combined with human body feeding experiments that the composition is safe and effective for maintaining a healthy blood sugar level and long-term use, and proves from the network pharmacology and molecular docking levels that the composition does not act through a single target, but embodies the synergistic action characteristics of traditional Chinese medicine'multi-component-multi-target-multi-pathway'.

Description

Technical Field

[0001] This invention belongs to the field of health food technology, specifically relating to a composition containing Litsea cubeba extract, its preparation method and application. Background Technology

[0002] Litsea cubeba ( Lithocarpus litseifolius (Hance) Chun) belongs to the genus *Acta* of the family Fagaceae. Lithocarpus Litsea cubeba is an evergreen tree whose leaves contain dihydrochalcone compounds, giving them a sweet taste that can be used as a tea substitute, also known as sweet tea. Modern pharmacological research shows that phlorizin, a dihydrochalcone compound in Litsea cubeba leaves, can inhibit glucose reabsorption in the kidneys, thereby controlling blood sugar and intervening in the occurrence and development of diabetes and kidney disease. It can also promote glucose secretion and lower fasting and postprandial blood glucose levels without causing hypoglycemia. Phlorizin, trifolin, and phloretin, dihydrochalcone compounds, can prevent and improve diabetic complications, and their anti-diabetic effects are receiving increasing attention. In 2017, Litsea cubeba was approved by the National Health and Family Planning Commission as a new food resource raw material.

[0003] Litsea cubeba has a long history of use and significant medicinal effects. However, because it is not a traditional medicinal plant, it is not included in the Chinese Pharmacopoeia. Therefore, research on multi-component, multi-target compound formulations containing Litsea cubeba is still in its early stages and lacks pharmacodynamic evidence at the systems biology level. The mechanism by which it synergistically regulates glucose and lipid metabolism, insulin signaling pathways, and chronic low-grade inflammatory networks with other plant extracts or micronutrients has not been elucidated. Existing technologies also do not provide reproducible PK-PD association data, making it difficult to meet the requirements of evidence-based medicine for the development of compound formulations with clear mechanisms and definite efficacy.

[0004] Therefore, current technologies cannot provide a traditional Chinese medicine compound preparation with a clear mechanism, definite efficacy, repeatable verification, and compliance with international evidence-based medicine standards for the prevention and treatment of diabetes and its complications. Summary of the Invention

[0005] In view of this, in order to prevent the occurrence of acute metabolic disorders, prevent or delay the occurrence and development of complications, and improve the quality of life, the present invention addresses the problems of the prior art by providing a composition containing Litsea cubeba extract with a clear mechanism, safe and effective and suitable for long-term use, as well as its preparation method and application.

[0006] The first objective of this invention is to provide a composition comprising an extract of Litsea cubeba. To achieve the above objective, this invention employs the following technical solution:

[0007] A composition comprising an extract of Litsea cubeba, comprising the following raw materials in parts by weight: 60-110 parts of American ginseng extract, 80-130 parts of mulberry leaf extract, 70-140 parts of Litsea cubeba extract, 150-300 parts of Astragalus membranaceus extract, and 2-15 parts of chromium-enriched yeast.

[0008] Furthermore, the composition comprises the following raw materials in parts by weight: 70-105 parts of American ginseng extract, 85-125 parts of mulberry leaf extract, 80-130 parts of Litsea cubeba extract, 165-250 parts of Astragalus membranaceus extract, and 4-10 parts of chromium-enriched yeast.

[0009] In the preferred embodiment, the composition comprises the following raw materials in parts by weight: 73.0-102.2 parts of American ginseng extract, 89.0-124.6 parts of mulberry leaf extract, 84.5-126.7 parts of Litsea cubeba extract, 166.4-249.6 parts of Astragalus membranaceus extract, and 4-10 parts of chromium-enriched yeast.

[0010] Furthermore, the composition also includes excipients, including one or more of soluble starch, maltodextrin, and dextrin.

[0011] It is worth noting that in the composition containing Litsea cubeba extract described in this invention, the four extracts have high viscosity and strong hygroscopicity, and moisture-proofing is achieved by adding one or more excipients. The soluble starch, maltodextrin, and dextrin specified in this invention have good flowability or moisture-proofing properties, and the preferred mass ratio of the raw material mixture to the excipients is 1:1.07.

[0012] Furthermore, the American ginseng extract, mulberry leaf extract, Litsea cubeba extract, and Astragalus membranaceus extract are all water extracts.

[0013] It is worth noting that the chromium-enriched yeast used in this invention complies with the provisions of GB 14880-2012 "National Food Safety Standard for Chromium as a Nutritional Fortifier".

[0014] Litsea cubeba extract, produced through processes including pulverization, water extraction, filtration, concentration, drying, pulverization again, sieving, and packaging, has an auxiliary effect in lowering blood sugar. Current research has demonstrated that both ethyl acetate and aqueous extracts of Litsea cubeba can effectively reduce blood glucose and MDA levels in mouse serum, and can restore CAT levels in mouse serum, exhibiting a certain regulatory and protective effect in diabetic mice.

[0015] American ginseng extract is produced through main processes including pulverization, water extraction, filtration, concentration, drying, pulverization again, sieving, and packaging. It has an auxiliary effect in lowering blood sugar. The American ginseng polysaccharides it contains can significantly regulate the levels of triglycerides, total cholesterol, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol in mouse serum, and can be used as an adjunct treatment for diabetes.

[0016] Mulberry leaf extract is produced through processes such as pulverization, water extraction, filtration, concentration, drying, pulverization, sieving, and packaging. It has the effect of assisting in lowering blood sugar. Mulberry leaf extract has a certain promoting effect on α-amylase, a significant inhibitory effect on α-glucosidase, and varying degrees of inhibitory effect on sucrase.

[0017] Astragalus extract is produced through processes including pulverization, water extraction, filtration, concentration, drying, pulverization again, sieving, and packaging. Studies have shown that the α-glucosidase inhibitor in Astragalus is a reversible, non-competitive inhibitor; it can lower blood glucose levels in normal mice, and at a concentration of 80 mg / kg... -1 At the optimal dose, blood glucose levels are lowest, and it can significantly improve glucose tolerance in mice; Astragalus water extract contains α-glucosidase inhibitors, which can be used to treat diabetes.

[0018] Therefore, the composition containing Litsea cubeba extract disclosed in this invention uses Litsea cubeba extract, American ginseng extract, mulberry leaf extract, astragalus extract, and chromium-enriched yeast as main raw materials. The combination of Litsea cubeba and mulberry leaf controls blood glucose peaks, creating a more stable blood glucose environment for yeast chromium and American ginseng / astragalus, allowing them to better exert their effects in improving insulin sensitivity and protecting cells. This invention embodies the synergistic effect of traditional Chinese medicine's "multi-component, multi-target, multi-pathway" approach, exhibiting comprehensive effects in improving insulin sensitivity, reducing metabolic inflammation and oxidative stress, and protecting target organs, thereby delaying the progression of diabetes and its complications.

[0019] A second objective of this invention is to provide a method for preparing the composition containing Litsea cubeba extract as described above. To achieve the above objective, this invention employs the following technical solution: A method for preparing a composition containing Litsea cubeba extract: Litsea cubeba extract, American ginseng extract, mulberry leaf extract, and astragalus extract are weighed in a certain proportion and mixed evenly. Chromium-enriched yeast is then added and mixed evenly with the mixed extract in an equal increment. Excipients are then added and premixed for 30 minutes until evenly mixed to obtain the composition.

[0020] Furthermore, it also includes: (1) Add 85% edible alcohol to the composition to make soft material, the amount of which is about 25% to 30% of the composition mass, and granulate it through a 14-mesh sieve to obtain wet granules; (2) Take the wet granules and dry them at 50℃~60℃, controlling the moisture content to about 5%, and granulate them through a 14-mesh sieve to obtain dry granules.

[0021] It is worth noting that the content of phlorizin and trifolin in the extract of Litsea cubeba of the present invention is ≥20g / 100g.

[0022] A third objective of this invention is to provide an application of the composition comprising Litsea cubeba extract as described above. To achieve the above objectives, this invention employs the following technical solution: The application of a composition containing Litsea cubeba extract in the preparation of pharmaceuticals and health products for the prevention and improvement of diabetic complications.

[0023] Existing research indicates that the development of diabetes and its complications is caused by a complex pathological network driven by multiple factors, including insulin secretion defects, insulin resistance, chronic low-grade inflammation, oxidative stress, and microcirculatory disturbances. Given the significant "multi-factor, multi-effect, and non-linear" characteristics of this network, single herbal medicines or single active ingredients are unlikely to simultaneously regulate key nodes within the network at sufficiently safe dosages, thus limiting their practical effectiveness.

[0024] Traditional Chinese medicine compound preparations based on multiple active ingredients exhibit additive effects of multiple components targeting a single point, synergistic effects of multiple components targeting multiple points, and toxicity dispersion effects. These contribute to restoring blood glucose homeostasis and have mild effects with few side effects, achieving good clinical efficacy in the long-term treatment of diabetes. However, current formulation designs lack evidence based on systems biology, resulting in unclear pharmacodynamic synergistic or antagonistic relationships among the various medicinal materials.

[0025] This invention demonstrates, from the perspectives of network pharmacology and molecular docking, that the composition containing Litsea cubeba disclosed in this invention does not act through a single target, but rather embodies the synergistic effect of traditional Chinese medicine characterized by "multiple components, multiple targets, and multiple pathways." Its hypoglycemic mechanism may use PPARG as a key regulatory hub, synergistically regulating the AGE-RAGE signaling pathway (targeting complications) and the endocrine resistance pathway (targeting core pathology) to achieve a comprehensive effect of improving insulin sensitivity, reducing metabolic inflammation and oxidative stress, and protecting target organs, thereby delaying the progression of diabetes and its complications. Compared with existing technologies, this invention follows the modern concept of "multi-target, multi-pathway synergistic effect" in its compound formulation design. Specifically, Litsea cubeba / mulberry leaf controls blood glucose peaks, creating a more stable blood glucose environment for yeast-rich chromium and American ginseng / Astragalus, allowing them to better exert their effects in improving insulin sensitivity and protecting cells. The overall internal environment improved by American ginseng / Astragalus may also provide comprehensive benefits through regulating blood lipids (mulberry leaf, Litsea cubeba, Astragalus), combating fatigue (American ginseng), and enhancing immunity (Astragalus, American ginseng), making it suitable for individuals with metabolic syndrome. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0027] Figure 1 This is a Venn diagram of the compound target gene and the diabetes target gene disclosed in Example 2 of the present invention.

[0028] Figure 2 This is a String network diagram of the intersection target genes in Embodiment 2 of the present invention.

[0029] Figure 3 This is a PPI network diagram of the target gene in Example 2 of the present invention.

[0030] Figure 4 This is a compound-disease-target network diagram in Example 2 of the present invention.

[0031] Figure 5 This is a schematic diagram of the GO enrichment analysis results in Embodiment 2 of the present invention.

[0032] Figure 6 This is the KEGG analysis result in Example 2 of the present invention.

[0033] Figure 7 The results show the visualization of PPARG and various compounds in Example 2 of this invention.

[0034] Figure 8 The effect of drug-containing serum and blank serum on the activity of 3T3-L1 cells in Example 3 of the present invention is shown. Among them, 1-blank serum, 2-10: the numbers are consistent with the orthogonal experiment numbers.

[0035] Figure 9 This is the result of the Oil Red O staining experiment used in Example 3 of the present invention to verify the induced differentiation experiment.

[0036] Figure 10 The glucose level in the cell supernatant of Example 3 of this invention.

[0037] Figure 11 This is the result of establishing the insulin resistance model in Example 3 of the present invention.

[0038] Figure 12 Example 3 of this invention illustrates the effect of the test substance on glucose uptake levels in insulin-resistant 3T3-L1 cells. Detailed Implementation

[0039] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] The term "embodiment" used herein, as an example, is not necessarily to be construed as superior to or better than other embodiments. Performance testing in the embodiments of this application, unless otherwise specified, employs conventional testing methods in the art. It should be understood that the terminology used in this application is merely for describing particular implementations and is not intended to limit the scope of this disclosure.

[0041] Unless otherwise stated, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; other experimental methods and technical means not specifically mentioned herein refer to experimental methods and technical means commonly used by one of ordinary skill in the art.

[0042] To better illustrate the content of this application, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that this application can be implemented even without certain specific details. In the embodiments, some methods, means, instruments, and devices well-known to those skilled in the art are not described in detail in order to highlight the main points of this application.

[0043] Without conflict, the technical features disclosed in the embodiments of this application can be combined arbitrarily, and the resulting technical solution belongs to the content disclosed in the embodiments of this application.

[0044] This invention discloses a composition containing Litsea cubeba extract, its preparation method, and its application, belonging to the field of health food technology. The composition disclosed in this invention uses Litsea cubeba extract, American ginseng extract, mulberry leaf extract, Astragalus extract, and chromium-enriched yeast as main raw materials. Animal experiments have demonstrated that it helps maintain healthy blood glucose levels. Furthermore, network pharmacology and molecular docking have proven that the composition does not act through a single target, but rather embodies the synergistic effect of traditional Chinese medicine's "multi-component-multi-target-multi-pathway" approach. Its hypoglycemic mechanism uses PPARG as a key pivot, synergistically regulating the AGE-RAGE signaling pathway (targeting complications) and the endocrine resistance pathway (targeting core pathology) to achieve a comprehensive effect of improving insulin sensitivity, reducing metabolic inflammation and oxidative stress, and protecting target organs, thereby delaying the progression of diabetes and its complications.

[0045] To better understand the present invention, the following embodiments are provided for further detailed description of the present invention, but they should not be construed as limiting the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above-described invention are also considered to fall within the protection scope of the present invention.

[0046] Example 1 Pilot-scale preparation of compositions containing Litsea cubeba extract (using granules as an example) Four pilot production runs were conducted on June 1, 2, 3, and August 10, 2023, respectively. These pilot production runs validated the process parameters used in the small-scale trials. The batch numbers were 20230601, 20230602, 20230603, and 20230810 (parameters are shown in Table 1). The validation results showed a yield rate of over 90%, indicating that the process is stable and controllable, the product meets requirements, and it is suitable for industrial-scale production.

[0047] Table 1 Pilot Production Data

[0048] 3.374 kg was taken out for toxicological function testing, and the remaining 21.70 kg was used to make the finished product.

[0049] Example 2 By searching TCMSP and specifying OB≥30% and DL≥0.18, 9 effective components of American ginseng were identified, corresponding to 79 targets; 25 effective components of mulberry leaf were identified, corresponding to 500 targets; 17 effective components of astragalus were identified, corresponding to 462 targets; and 47 known components of Litsea cubeba were identified through literature review, corresponding to 2071 targets. After merging and deduplicating, a total of 589 corresponding targets were collected and entered into the Uniprot database to obtain standard gene names. After merging all database targets and removing duplicates, a total of 681 disease targets were obtained.

[0050] The 589 target genes of the obtained traditional Chinese medicine compound were compared with the diabetes target genes, and the repetition values ​​were taken to obtain 169 intersection genes. A Venn diagram was then constructed, as shown in [reference needed]. Figure 1 The 169 intersecting target genes were imported into String to obtain the PPI network diagram, as shown below. Figure 2 , 3The nodes in the diagram represent target proteins, and the lines represent the relationships between proteins. There are a total of 169 nodes and 4037 edges, with an average node degree of 47.8. A TSV file was exported from the database and imported into Cytoscape for network analysis. Node size and color were selected based on Degree values, ranging from small to large and from dark to light; edges were also selected based on Combined Score values, ranging from thin to thick and from dark to light. Core target proteins with a Degree value ≥ 116 were arranged in a central circle, while other target proteins were arranged in a peripheral circle. Figure 3 Its core targets are AKT1, ALB, IL6, TNF, and PPARG.

[0051] PPARG, or peroxisome proliferator-activated receptor gamma, is a member of the nuclear hormone receptor superfamily and primarily functions as a key regulator of adipocyte differentiation and energy storage. Once activated by its ligand, it regulates the expression of a series of genes involved in lipid metabolism, glucose stability, and insulin sensitivity. Its applications are mainly in type 2 diabetes and metabolic syndrome. Albumin (ALB) is the most abundant protein in human blood plasma, synthesized by the liver. Its main functions include maintaining plasma colloid osmotic pressure, transporting endogenous (such as bilirubin and fatty acids) and exogenous substances (such as many drugs), and antioxidation. It is commonly found in cases of severely impaired liver function (such as cirrhosis and severe hepatitis), malnutrition, nephrotic syndrome (loss in urine), and chronic wasting diseases (such as advanced cancer). Its applications are mainly in liver disease, kidney disease, and malnutrition.

[0052] Using Cytoscape software, a compound-disease-target network was constructed. (See...) Figure 4 Topological analysis revealed that its core targets were: quercetin (Degree = 309), kaempferol (Degree = 127), apigenin, tricin, fisetin, and luteolin (all Degree = 101).

[0053] Quercetin: Found in mulberry leaves, litsea cubeba leaves, and astragalus, quercetin is a widely distributed natural flavonoid compound with pharmacological effects including anti-obesity, hypoglycemic, antibacterial, anti-inflammatory, and antiviral properties. Studies have shown that it can intervene in the pathophysiological process of diabetes at multiple stages. [3]① Prevention and improvement of insulin resistance: In target tissues such as muscle, fat, and liver, quercetin repairs damaged insulin signaling through its anti-inflammatory effects and activation of the AMPK / PI3K pathway, enabling cells to respond more effectively to insulin and thus lowering blood sugar. Multiple animal studies and some human clinical trials have shown that quercetin supplementation can significantly reduce fasting blood glucose, fasting insulin, and insulin resistance index. ② Direct hypoglycemic effect: In addition to improving insulin sensitivity, it can also inhibit intestinal α-glucosidase (similar to acarbose), inhibit hepatic gluconeogenesis, and promote glucose utilization in peripheral tissues. Meta-analysis of patients with type 2 diabetes showed that quercetin supplementation can effectively reduce glycated hemoglobin. ③ Prevention Treatment of chronic complications of diabetes: Quercetin may have a greater advantage over simple hypoglycemic drugs in this area because it directly targets the root causes of complications—oxidative stress and inflammation. For example, quercetin alleviates streptococcal-induced retinopathy in rats by downregulating monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinase-9 (MMP-9), and vascular endothelial growth factor (VEGF); quercetin treatment reduces diabetic nephropathy in hypercholesterolemia mice by lowering blood glucose and triglyceride levels; quercetin also has neuroprotective effects in the cecum of experimentally diabetic rats; ④ Protection of pancreatic β-cell function: In the diabetic state, β-cells are continuously attacked by glucose-lipid toxicity and inflammation. The protective effect of quercetin helps maintain their ability to secrete insulin and slows disease progression. Current status: Clinical trials on the effects of quercetin in patients with type 2 diabetes are limited. A single dose of quercetin (400 mg) inhibits α-glucosidase activity and reduces postprandial hyperglycemia in T2DM. In patients with metabolic disorders, quercetin at a daily dose of 500 mg / day reduced plasma glucose levels. Furthermore, oral quercetin (250 mg / day) improved the antioxidant status of patients with type 2 diabetes mellitus (T2DM). Daily quercetin intake (20.9 ± 2.32 mg / day) reduced the prevalence of T2DM in the Chinese population. These differences in clinical trial results may stem from dose variations or intervention duration, which could be key factors determining the clinical efficacy of quercetin.

[0054] Kaempferol: This component is found in mulberry leaves, Litsea cubeba leaves, and Astragalus membranaceus. It has anti-inflammatory, antioxidant, hypoglycemic, anti-tumor, anti-atherosclerotic, and lipid-lowering effects. It plays a role in multiple aspects of diabetes management: ① Improving insulin resistance and lowering blood sugar (core function): By simultaneously "opening up" (activating AMPK to promote glucose uptake) and "reducing" (inhibiting hepatic gluconeogenesis to reduce glucose output), and enhancing insulin signaling, it effectively reduces peripheral insulin resistance. This is key to its prevention and treatment of type 2 diabetes. Numerous animal experiments have shown that kaempferol can significantly reduce fasting blood glucose, fasting insulin, and insulin resistance index in obese or diabetic models. Its hypoglycemic effect is similar to that of classic metformin (which also relies on AMPK activation); ② Preventing and treating diabetic complications (key advantage): Kaempferol's powerful antioxidant and anti-inflammatory capabilities make it highly promising in preventing and treating chronic diabetic complications. Diabetic nephropathy: Inhibits renal mesangial cell proliferation and fibrosis, reduces renal oxidative damage and inflammation, and decreases proteinuria; Diabetic cardiomyopathy: Upregulates SIRT1, inhibits NF-κB nuclear translocation, and activates nuclear factor E2-related factors, thereby inhibiting diabetic myocardial inflammation and oxidative stress, and can reduce AGE-RAGE / MAPK-induced oxidative stress and inflammation to alleviate myocardial ischemia / reperfusion injury; Diabetic retinopathy: Inhibits retinal vascular endothelial cell apoptosis and pathological angiogenesis; Diabetic macrovascular disease: Delays the progression of atherosclerosis by protecting the endothelium, reducing inflammation, and regulating blood lipids; ③ Regulating lipid metabolism and improving non-alcoholic fatty liver: Diabetes is often accompanied by lipid metabolism disorders. Kaempferol can promote fatty acid oxidation and reduce triglyceride accumulation in the liver and blood, which has a positive effect on improving non-alcoholic fatty liver disease associated with diabetes. ④ Potential protective effects: Some studies have shown that kaempferol may regulate postprandial blood glucose to some extent by inhibiting intestinal α-glucosidase.

[0055] Apigenin: This component, found in Litsea cubeba, is a natural bioactive flavonoid widely present in medicinal plants, functional foods, vegetables, and fruits. It possesses properties that alleviate metabolic diseases, have anti-inflammatory, antioxidant, and anti-obesity effects, as well as anti-proliferative and anti-cancer activities. Its relationship with diabetes can be summarized as acting as both an "inflammation extinguisher" and a "metabolic regulator." By potently inhibiting the NF-κB inflammatory pathway and activating the AMPK / Nrf2 protective pathway, it achieves: alleviating insulin resistance at its source, improving glycemic control, and systematically defending against oxidative damage and inflammation, thereby effectively preventing and delaying various diabetic complications. It uniquely demonstrates the potential to protect and even potentially repair pancreatic β-cells. Apigenin plays a role in multiple aspects of diabetes management: ① Fundamentally improving insulin resistance: Its powerful anti-inflammatory effect directly reduces the inflammatory state of adipose tissue, liver, and muscles, and repairs the insulin signaling pathway. Meanwhile, activating AMPK provides an insulin-independent hypoglycemic pathway. In diet-induced obesity and diabetic animal models, apigenin supplementation can significantly reduce fasting blood glucose and fasting insulin levels, and improve insulin resistance index, sometimes with effects comparable to metformin; ② Prevention and treatment of diabetic complications: Diabetic nephropathy: Reduces glomerular sclerosis, renal oxidative stress, inflammatory cell infiltration and fibrosis in STZ-induced diabetic nephropathy rats, reduces proteinuria, and protects renal function; Diabetic neuropathy: Protects nerve cells, improves nerve conduction function and pain; Diabetic cardiovascular disease: Improves vascular endothelial function, inhibits atherosclerotic plaque formation, reduces myocardial HIF-1α expression and upregulates myocardial PPARγ and its target genes—glycerol-3-phosphoacyltransferase gene (GPAT) and glucose transporter (GLUT-4) expression, and improves abnormal myocardial glucose and lipid metabolism; Diabetic retinopathy: Inhibits inflammatory response and abnormal angiogenesis in retinal vessels; ③ Regulation of lipid metabolism and improvement of non-alcoholic fatty liver: Diabetes is often accompanied by dyslipidemia and fatty liver. Apigenin can promote fatty acid oxidation, reduce lipid accumulation in the liver, and improve liver insulin sensitivity, and has a good effect on improving non-alcoholic fatty liver disease (NAFLD) associated with diabetes; ④ Potential β-cell protection and functional regeneration: In addition to protecting existing β-cells, it may induce β-cell regeneration (although it is mainly in the preclinical research stage), which brings new hope for repairing the core defects of diabetes.

[0056] Alfalfa extract: This component, also known as "strychnos nucifera extract," is found in Litsea cubeba. Alfalfa extract has shown multifaceted benefits in the management of diabetes and its complications. Prevention and treatment of diabetic complications (important research direction): Diabetic cardiomyopathy: It has a protective effect against hyperglycemia-induced cardiac injury in a DCM cell model. By reducing oxidative stress and inflammation, and inhibiting the TLR4-MYD88-NF-κB pathway, the value of alfalfa extract as a novel therapy for the management of diabetic cardiomyopathy is highlighted; Diabetic retinopathy: By regulating Sestrin 2 / Nrf 2 signaling, it inhibits oxidative stress and angiogenesis, and alleviates inflammation, thereby reducing diabetic retinopathy. Currently, there is limited research on alfalfa extract for diabetes.

[0057] Rhubarb: This component, found in Litsea cubeba, has transcended traditional hypoglycemic and antioxidant effects in diabetes management, entering a new dimension of "anti-aging therapy." ① Improvement of insulin resistance and metabolic parameters: As a traditional drug for long-term treatment of diabetes and metabolic diseases, it exhibits insulin-promoting effects. Through anti-inflammatory action and the removal of senescent adipocytes and muscle cells, it directly improves the microenvironment of these insulin target tissues, thereby restoring insulin sensitivity. In obese and diabetic animal models, rhubarb effectively reduces fasting blood glucose, insulin levels, and insulin resistance index, and alleviates body weight and hepatic steatosis. ② Prevention and treatment of diabetic complications (a highly promising direction): Its senolytic (removal of senescent cells) and neuroprotective properties make it a promising candidate for the prevention and treatment of complications. Diabetic neuropathic pain: It exerts an anti-pain effect on diabetic neuropathic pain in mice through antioxidant mechanisms and binding to spinal GABAA receptors. Diabetic nephropathy: Rhubarb can alleviate hyperglycemia-induced podocyte damage and STZ-induced diabetic nephropathy in mice by restoring the autophagy-mediated CDKN1B / P70S6K pathway and inhibiting the NLRP3 inflammasome. Diabetic neuropathy: This is a major highlight of rosin research. It can prevent oxidative stress and neuroinflammation caused by diabetes-related peripheral nerve damage by inhibiting NF-κB and positively regulating Nrf2; Diabetic encephalopathy / cognitive impairment: It improves diabetes-related learning and memory decline, protects hippocampal neurons, and prevents high glucose-induced neurotoxicity by activating the PI3K / Akt / CREB pathway, exhibiting a strong neuroprotective effect; Diabetic cardiomyopathy: It can inhibit oxidative stress in the heart of diabetic rats, prevent inflammation and apoptosis, and enhance antioxidant defense capabilities, thereby alleviating the development of diabetic cardiomyopathy; Potential protection of pancreatic β-cell function: It reduces the glucolipotoxic damage of β-cells through antioxidant and anti-apoptotic effects.

[0058] Luteolin: This component, found in Litsea cubeba, exhibits unique benefits in diabetes management, particularly in its potent anti-inflammatory and anti-fibrotic effects. ① Strongly improves insulin resistance (core efficacy): Its powerful anti-inflammatory effect directly and fundamentally reduces inflammation in adipose tissue and throughout the body, repairing insulin signaling. Simultaneously, by reversing hyperlipidemia, oxidative stress, and pro-inflammatory states, luteolin significantly reduces fasting blood glucose, fasting insulin, and insulin resistance index in a high-fat diet-induced obese diabetic animal model, demonstrating its anti-diabetic activity. ② Excellent potential for preventing and treating diabetic complications (especially kidney and liver disease): The anti-inflammatory and anti-fibrotic properties of luteolin give it a unique advantage in preventing and treating organ fibrosis complications. Diabetic nephropathy: This is one of the most extensively studied areas for luteolin. It significantly reduces proteinuria and protects kidney function. Mechanism is clear: By inhibiting the NF-κB inflammatory pathway and the TGF-β1 / Smad fibrotic pathway, it works in two ways to reduce kidney inflammation, mesangial matrix proliferation, and renal interstitial fibrosis. Nonalcoholic fatty liver disease and liver fibrosis: Through the interaction between the liver and adipose tissue, it improves hepatic lipid deposition, hepatic steatosis, and insulin resistance. Diabetic neuropathy and vascular complications: Through anti-inflammatory and antioxidant effects, it protects nerve and vascular endothelial function. Diabetic vascular dysfunction: Luteolin has been shown to improve endothelial dysfunction in type 2 diabetes by improving lipid index, lipid profile, fasting blood glucose levels, glucose and insulin tolerance tests, and endothelial function studies, demonstrating its therapeutic potential for treating type 2 diabetes-related vascular complications. Potential pancreatic β-cell protective effects: By alleviating STZ-induced β-cell apoptosis, metabolic disorders, and oxidative stress, it may improve β-cell function and slow the progression of diabetes.

[0059] 169 genes with overlapping target sites were imported into DAVID for enrichment analysis of GO biological processes, GO molecular functions, GO cellular components, and the KEGG pathway. GO biological processes yielded 792 enrichment results, GO molecular functions yielded 199, and GO cellular components yielded 73, for a total of 1064 results in GO functional enrichment analysis. The top 15, 10, and 10 representative enrichment items were selected, and bar charts were created using an online plotting tool, as shown below. Figure 5The biological processes of GO mainly involve RNA polymerase II-mediated positive regulation of transcription, hypoxia stress response, negative regulation of apoptosis, response to exogenous stimuli, positive regulation of gene expression, nuclear receptor-mediated steroid hormone signaling pathway, and positive regulation of DNA template transcription. GO molecular functions are mainly related to nuclear receptors, enzyme binding, homologous protein binding, intranuclear steroid receptor activity, heme binding, steroid binding, DNA-binding transcription factor activity, sequence-specific DNA binding, estrogen response element binding, and transcription coactivator binding. GO cellular components mainly include membrane rafts, protein complexes, chromatin, RNA polymerase II transcriptional regulatory complex, cytoplasm, cytosol, nucleoplasm, and plasma membrane.

[0060] KEGG pathway enrichment results identified 179 relevant pathways. A bubble chart was created from 15 representative pathways, primarily including: the AGE-RAGE signaling pathway in diabetic complications, cancer pathways, lipids and atherosclerosis, chemical carcinogenesis-receptor activation, fluid shear stress and atherosclerosis, endocrine resistance, EGFR tyrosine kinase inhibitor resistance, and the TNF signaling pathway (see...). Figure 6 ).

[0061] Among them, the AGE-RAGE signaling pathway in diabetic complications reveals a core and persistent molecular mechanism by which hyperglycemia leads to organ damage. The sustained activation of this pathway is a key driver in the occurrence and development of chronic diabetic complications such as retinopathy, nephropathy, neuropathy, and atherosclerosis. AGEs formation occurs when, under sustained hyperglycemic conditions, reducing sugars such as glucose undergo non-enzymatic reactions with the amino groups of proteins, lipids, and nucleic acids, ultimately forming advanced glycation end products (AGEs). This is a slow, irreversible cumulative process. RAGE activation occurs when AGEs, acting as ligands, bind to AGE receptors on the cell membrane. RAGE is a pattern recognition receptor whose activation triggers strong and persistent inflammatory and oxidative stress signals. After AGE-RAGE binding, it mainly activates key transcription factors such as NF-κB, leading to: the massive production of pro-inflammatory cytokines, reactive oxygen species bursts causing oxidative damage, expression of pro-fibrotic factors promoting tissue fibrosis, vascular endothelial dysfunction, increased permeability, and a tendency to form thrombi. Once formed, AGEs accumulate in tissues for a long time and continue to cause harm, leading to complications such as diabetic nephropathy, diabetic retinopathy, and diabetic neuropathy. This provides a mechanistic clue to explain the potential value of the compound disclosed in this invention in improving diabetic vascular complications (such as nephropathy and retinopathy).

[0062] Endocrine resistance refers to a decreased sensitivity of the body to specific hormones, requiring higher than normal levels to produce normal physiological effects. This is the common pathological basis of many metabolic and endocrine diseases, rather than the disease itself. The two most common and important types are insulin resistance and leptin resistance. Insulin resistance is the most prevalent and far-reaching type of endocrine resistance, a central link in metabolic syndrome: it connects obesity, hypertension, dyslipidemia, and hyperglycemia, and is a fundamental precursor to type 2 diabetes. To compensate, pancreatic β-cells over-secrete insulin, leading to hyperinsulinemia. When β-cell function eventually becomes decompensated, overt diabetes occurs. Leptin resistance is caused by the hypothalamus's insensitivity to leptin signals. This further supports the systemic effect of the compound formula disclosed in this invention in improving the body's resistance to insulin and other metabolic hormones, corroborating the function of the PPARG target and forming a complete mechanism of action from the molecular to the pathway.

[0063] Based on the above results, the key components were selected as follows: quercetin, kaempferol, Apigenin, Tricin, Fisetin, and Luteolin; the key targets were: AKT1, ALB, IL6, PPARG, and TNF. Molecular docking was performed in AutoDock. The binding energy unit is kcal / mol. Binding energy is a concept of "free energy," which describes the energy change of the entire process of ligand and acceptor binding to form a complex. When it is less than 0, it indicates that the binding process is spontaneous and energy-favorable. The larger the absolute value of the complex number, the stronger and more stable the binding is generally. The binding energies are shown in Table 2. The red part represents the compounds, the yellow part represents the hydrogen bonds, and the combination of letters and numbers next to the hydrogen bonds is key to understanding the interactions. The remaining part represents the targets. AKT1, ALB, IL6, and TNF all bind to each compound, and PPARG binds strongly to each core compound. Figure 7 PPARG, a key nuclear receptor regulating lipid metabolism, glucose homeostasis, and insulin sensitivity, can promote adipocyte differentiation, increase glucose uptake by adipose tissue, and improve systemic insulin sensitivity upon activation. Therefore, the American ginseng, mulberry leaf, ginger, and angelica compound disclosed in this invention may improve insulin resistance, a core pathological aspect of type 2 diabetes, from an upstream perspective by directly activating or regulating PPARG.

[0064] Table 2 Binding Energy

[0065] Thus, the present invention proves from the aspects of network pharmacology and molecular docking that the composition containing Lithocarpus litseifolius (Hance) Chun disclosed in the present invention does not act through a single target, but reflects the synergistic effect characteristics of traditional Chinese medicine of "multiple components - multiple targets - multiple pathways". Its hypoglycemic mechanism may take PPARG as the key action hub, and through the coordinated regulation of the AGE-RAGE signaling pathway (for complications) and the endocrine resistance pathway (for the core pathology), achieve the comprehensive effects of improving insulin sensitivity, reducing metabolic inflammation and oxidative stress, and protecting target organs, thereby delaying the progression of diabetes and its complications, providing a clear direction and hypothesis basis for subsequent in vitro and in vivo experimental verification.

[0066] Example 3 1 Materials and Methods 1.1 Orthogonal Experiment Design Test substances: Medicated sera with various ratios of American ginseng, mulberry leaves, and Lithocarpus litseifolius (Hance) Chun; the compound levels are set as shown in Table 3.

[0067] Table 3 Compound Level Settings

[0068] There are a total of 4 factors above, with 3 levels for each factor. The most suitable orthogonal table is L9(34).

[0069] Table 4 Orthogonal Table L9(34)

[0070] According to the selected orthogonal table, substitute the levels of each factor to form 9 specific test formulas for this example (Table 5).

[0071] Table 5 Test Formulas

[0072] 1.2 Preparation of Medicated Serum 60 male SD rats, weighing 200±20 g (use license number: SYXK (Xiang) 2023-0003), were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd. and raised in the SPF-level barrier environment of Hunan An Sheng Mei Pharmaceutical Research Institute Co., Ltd. Maintained under standard laboratory conditions: room temperature 20.0 - 26.0 °C, relative humidity 40.0 - 70.0%. All operations in this study were strictly carried out in accordance with the relevant laws and regulations on the welfare and ethics of experimental animals in China and the relevant standard operating procedures of the Experimental Animal Welfare and Ethics Committee of Hunan An Sheng Mei Pharmaceutical Research Institute Co., Ltd. (ethical approval number: IACUC-XLH-2023-107). After one week of adaptive feeding, the rats were randomly divided into 10 groups: a normal control group and the above numbered groups, with 6 rats in each group. The adult daily dosage of the American ginseng, mulberry leaf, and Lithocarpus litseifolius (Hance) Chun granules after removing excipients is 6 g. According to the equivalent dose conversion, the prescription ratio dosing doses are all 6 0.018 / 0.2 = 0.54 g / kg. After administering the drug at 2.16 g / kg (four times the equivalent dose) via gavage for 14 consecutive days, blood was collected from the abdominal aorta, with at least three vacuum blood collection tubes per rat. The collected whole blood was allowed to stand at room temperature for 30-60 minutes, then centrifuged at 3000 rpm at 4 ℃ for 15 minutes. The supernatant was collected, aliquoted, and stored at -80 ℃ for later use.

[0073] 1.3 Cell Culture, Modeling, and Grouping Using 3T3-L1 mouse embryonic fibroblasts (Haixing Biotechnology, catalog number: TCM-C702) as the research subject, when the cell density reached 80%-90%, they were differentiated into 3T3-L1 adipocytes using the 3T3-L1 adipocyte differentiation induction medium kit (Pronosa, catalog number: PD-031). Adipocyte differentiation induction medium A was used for 3 days, followed by replacement with adipocyte differentiation induction medium B for 1 day. This process was repeated 4 times, alternating between 3 days of medium A and 1 day of medium B. Oil Red O staining was used to verify successful differentiation; the cells were considered to be filled with red lipid droplets. After successful differentiation, the cells were cultured in high-glucose DMEM basal medium with a low concentration of 1 µg / mL insulin and divided into two groups: a normal control group (adipocytes + 10% blank serum (number 1)) and an insulin resistance model (adipocytes + 1 µM insulin). 1 (Pronox, catalog number: PB180432) + 10% blank serum), each treatment group (adipocytes + 1µM insulin + 10% of each drug-containing serum (numbers 2-10 correspond to the orthogonal test numbers above)), positive group (adipocytes + 1µM insulin + 1mM metformin (Ambeed, catalog number: A270611) + 10% blank serum).

[0074] 1.4 Effect of different formulation ratios on cell viability as determined by CCK-8 assay The effect of different concentrations of drug-containing serum on cell viability was investigated using CCK8 assay: Cells were seeded in 96-well plates, and after adhesion, the medium was replaced with serum containing different concentrations (e.g., 0%, 2.5%, 5%, 10%, 20%, 40%) of the prescribed drug-containing serum. After co-incubation for 24 h, CCK8 reagent was added, and the OD value was measured at 450 nm after incubation for 1–4 h. The safe concentration of different drug-containing serum formulations for non-toxicity was determined for subsequent experiments.

[0075] 1.5 Effect of different formulation ratios on residual glucose in cell supernatant using the GOD-POD colorimetric method Induced mature adipocytes were seeded in 6-well plates and grouped according to the experimental design. Before intervention, the culture medium was replaced with serum-free DMEM basal medium containing 1 µg / mL insulin. Each group was pre-incubated with the corresponding concentration of drug-containing serum, while the normal control group, model group, and positive control group were supplemented with blank serum. After 24 hours of pre-incubation, all groups except the normal control group (including the positive control group) were incubated with 1 µM insulin for another 24 hours (the positive control group was co-incubated with insulin for 12 hours). After intervention, the cell culture supernatant was collected, and the glucose assay kit (Elabscience, E-BC-K234-M) was strictly followed. Higher glucose consumption → less glucose remaining in the culture medium → indicates stronger glucose uptake and utilization by the cells and better insulin sensitivity. OD values ​​were measured at 505 nm using a microplate reader.

[0076] 1.6 Effect of different formulation ratios on cellular glucose uptake levels by 2-NBDG flow cytometry After the above intervention was completed, the culture medium was discarded, the cells were washed twice with PBS, and then starved for 2 hours in sugar-free DMEM culture medium. The cells were then treated with the 2-NBDG glucose uptake kit (Elabscience, catalog number: E-CK-A441), and flow cytometry was performed. The FITC channel was selected, and the cells were processed using FlowJo software.

[0077] 1.7 Statistical Analysis All measurement data are expressed as mean ± standard deviation. The results are expressed as mean ± standard deviation (SD), and statistical analysis was performed using SPSS 26.0 software and graphs were generated using GraphPad Prism 9 software. Data with a statistical significance level set at P < 0.05 were analyzed using one-way ANOVA and post-hoc LSD statistical analysis. Nonparametric rank-sum tests were used for non-normally distributed or unequal variance (P < 0.05) data.

[0078] 2. Experimental Results 2.1 Effects of the test substance on the viability of 3T3-L1 cells The effects of different concentrations (0%, 2.5%, 5%, 10%, 20%, 40%) of drug-containing serum and blank serum on the viability of 3T3-L1 cells were detected using a CCK-8 assay. The results showed that cell viability did not significantly decrease at concentrations of the test substance at 10% and below, and the viability remained above 90%. However, when the concentration increased to 20%, cell viability began to show significant inhibition. Figure 8 The results showed that the test substance could be used at a safe concentration range of 10% in subsequent pharmacodynamic experiments.

[0079] 2.2 Oil Red O staining to verify differentiation into adipocytes Oil Red O staining was performed on both cells 14 days after induced differentiation and those without induced differentiation for comparison (see...). Figure 9 The cells that were induced to differentiate were found to be filled with orange-red lipid droplets, indicating that 3T3-L1 cells were successfully induced to differentiate into adipocytes, which can be used to establish a subsequent insulin resistance model.

[0080] 2.3 Effect of the test substance on the residual glucose content in the supernatant of insulin-resistant 3T3-L1 cells The results of the insulin resistance model induced in successfully differentiated cells are shown in the figure. Figure 10 Compared with the normal control, the residual glucose level in the model group was significantly increased, indicating that the cells' ability to take up and utilize glucose was weakened and the insulin resistance model was successfully established. After drug intervention, except for group 10, the residual glucose level in the other numbered groups was significantly reduced, and the ability to take up and utilize glucose was enhanced. Groups 5, 6, and 8 had the lowest residual glucose levels, which were close to those in the normal group. This indicates that the ratio of these three groups can effectively reduce the residual glucose level in 3T3-L1 differentiated cells under the insulin resistance model.

[0081] 2.4 Effect of the test substance on glucose uptake levels in insulin-resistant 3T3-L1 cells 2-NBDG, as a fluorescent glucose analog, directly reflects the cellular glucose uptake capacity through flow cytometry-measured fluorescence intensity (Mean Fluorescence Intensity, MFI). Results showed that compared to normal controls, the model group exhibited significantly decreased MFI, indicating weakened glucose uptake capacity. Intervention with the positive control drug metformin significantly increased MFI, indicating successful establishment of the insulin resistance model. Figure 11 .

[0082] After drug intervention, the MFI of group 7 was significantly increased and higher than that of other drug groups and the normal control group. Groups 6 and 9 were the next most significantly increased, with their MFI also higher than that of other drug groups. This indicates that groups 6, 7, and 9 can effectively enhance the glucose uptake capacity of insulin-resistant 3T3-L1 differentiated cells. Figure 12 .

[0083] 3. Experimental Conclusions 1. Safety evaluation: The drug-containing serum showed no significant toxicity to 3T4-L1 cells at a concentration of ≤10%, indicating its biosafety for further studies.

[0084] 2. GOD-POD and 2-NBDG tests revealed that, except for the 10th formulation which showed no significant difference, the serum containing the drug and the positive control in the other formulations significantly reduced the amount of glucose remaining in the cell supernatant, effectively improving the cellular glucose uptake dysfunction caused by insulin resistance.

[0085] 3. Superior formula ratio: Through comprehensive comparison of GOD-POD and 2-NBDG, it is found that formula ratios 6 (A2B2C3D1), 7 (A2B3C1D2), and 9 (A3B2C1D3) have the best in vitro hypoglycemic effect and may be potential optimal formula ratios. Considering both, formula ratio A2B2C3D1 is the best among the three.

[0086] Example 4 1. Materials and Methods 1.1 Samples: The samples obtained in Example 1, with the recommended oral dose for humans being 10 g per day. Calculated based on an adult body weight of 60 kg, the equivalent dose is 0.167 g / kg·bw. Due to the relatively large recommended dose, the method of removing excipients is adopted. After removing excipients, the recommended oral dose for humans is 6 g per day. Calculated based on an adult body weight of 60 kg, the equivalent dose is 0.1 g / kg·bw.

[0087] 1.2 Experimental animals: 110 SPF-grade male ICR mice were provided by Changsha Tianqin Biotechnology Co., Ltd., with a body weight of 20 g - 24 g. The production license number for experimental animals is SCXK(Xiang)2022 - 0011. Among them, 90 animals were used to establish hyperglycemic models for measuring the effect of the sample on the blood glucose of hyperglycemic model animals, and 20 animals were used for the experiment on the effect of the sample on the blood glucose of normal animals.

[0088] 1.3 Experimental environmental conditions: It is a barrier environment. During the experiment, the environmental temperature is 22°C - 24°C, and the humidity is 50% - 54%. The license number for the use of experimental animals is SYXK(Xiang)2023 - 0005.

[0089] 1.4 Dose selection and sample preparation: According to the clear provisions in Article 2 of the "Technical Guidelines for the Function Testing and Evaluation of Health Foods (2023 Edition)", the low, medium, and high doses of the composition containing Lithocarpus litseifolius extract are set at 0.5 g / kg·bw, 1.0 g / kg·bw, and 3.0 g / kg·bw respectively (equivalent to 5, 10, and 30 times the human recommended dose). Take 5.0 g, 10.0 g, and 30.0 g of the sample and add purified water to 200 mL to prepare the required concentrations for low, medium, and high doses. The control group is given the same volume of purified water. Each group is gavaged once a day, with a gavage volume of 0.2 ml / 10 g·bw for 30 consecutive days.

[0090] 1.5 Main instruments, equipment, and reagents: Animal weighing balance; ONE TOUCH steady type blood glucose meter, with test strips provided by the same manufacturer.

[0091] 1.6 Experimental methods: 1.6.1 Experiment on reducing fasting blood glucose 1.6.1.1 Hyperglycemic model animals Fifteen mice were randomly selected and fasted for 3-5 hours. Fasting blood glucose levels were measured to determine the baseline blood glucose levels for this batch of animals. Subsequently, the mice were fasted for 24 hours (with free access to water) and then injected with alloxan (48.0 mg / kg bw) via the tail vein to induce the hyperglycemic model. Five days later, the mice were fasted for 5 hours, and blood glucose levels were measured. Animals with blood glucose levels of 10-25 mmol / L were considered successful hyperglycemic models. Forty successful hyperglycemic models were selected and randomly divided into one model control group and three dosage groups (with a difference between groups not exceeding 1.1 mmol / L). The dosage groups were given different concentrations of the test solution, while the model control group was given the solvent. Fasting blood glucose levels were measured after 5 hours of fasting for 30 consecutive days, and the blood glucose levels and percentage decrease in blood glucose were compared among the groups.

[0092] Percentage decrease in blood glucose = (Pre-experiment blood glucose level - Post-experiment blood glucose level) / Pre-experiment blood glucose level × 100%.

[0093] 1.6.1.2 Normal Animals Twenty mice were randomly divided into a control group and a test sample group (high-dose group) based on their blood glucose levels after a 5-hour fast. The high-dose group was given the test solution, while the control group was given the solvent. Fasting blood glucose levels were measured after a 5-hour fast for 30 consecutive days, and the blood glucose levels and percentage decrease in blood glucose were compared between the two groups.

[0094] Percentage decrease in blood glucose = (Pre-experiment blood glucose level - Post-experiment blood glucose level) / Pre-experiment blood glucose level × 100%.

[0095] 1.6.2 Glucose Tolerance Test At the end of the experiment, animals in each group were fasted for 5 hours, and fasting blood glucose levels were measured as the pre-glucose (0-hour) blood glucose levels. The dosage groups were given different concentrations of the test solution, while the model control group received the same volume of solvent. Glucose (2.0 g / kg body weight) was administered orally 15–20 minutes later. Blood glucose levels were measured at 0.5 and 2 hours after glucose administration to observe the model. Changes in blood glucose levels and area under the blood glucose curve at various time points after glucose administration in the control group and the test sample group.

[0096] Area under the blood glucose curve = 0.25 × (0-hour blood glucose value + 4 × 0.5-hour blood glucose value + 3 × 2-hour blood glucose value) 1.7 Experimental Data Processing Data transformation and statistical analysis were performed using Excel and SPSS software. When using SPSS for statistical comparisons, the data were first tested for homogeneity of variance. If the variances were homogeneous, one-way ANOVA was used for overall comparison, followed by Dunnett's method for pairwise comparisons between the means of multiple dose groups and a control group. If the variances were unequal, the original data underwent appropriate variable transformation to satisfy the homogeneity of variance test, and the transformed data was then used for statistical analysis. If homogeneity of variance was still not achieved after variable transformation, the rank-sum test was used for statistical analysis. If differences were found in the overall comparison, Tamhane's T² test, which does not require homogeneity of variance, was used for pairwise comparisons.

[0097] 1.8 Result Determination If either fasting blood glucose or glucose tolerance test is positive, and it has no effect on fasting blood glucose in normal animals, the animal experiment result for the blood glucose-lowering function of the test sample can be determined to be positive.

[0098] 2 Results 2.1 Effects of compositions containing Litsea cubeba extract on body weight in normal mice and hyperglycemic model mice The results are shown in Tables 6 and 7. It can be seen that the high dose of the composition containing Litsea cubeba extract had no significant effect on the body weight of normal mice, and the various doses of the composition containing Litsea cubeba extract had no significant effect on the body weight of hyperglycemic model mice at each time point (P>0.05).

[0099] Table 6. Effects of the samples on the body weight of normal mice ( x ± s , g)

[0100] Table 7. Effects of samples on body weight in hyperglycemic model mice ( x ± s , g)

[0101] 2.2 Effects of compositions containing Litsea cubeba extract on fasting blood glucose in normal mice See Table 8. Oral administration of high doses of the composition containing Litsea cubeba extract to mice for 30 days had no significant effect on fasting blood glucose levels or the percentage decrease in blood glucose in normal animals (P>0.05).

[0102] Table 8. Effects of the samples on fasting blood glucose in normal mice ( x ± s )

[0103] 2.3 Effects of compositions containing Litsea cubeba extract on fasting blood glucose in hyperglycemic model animals See Table 9. Mice were orally administered different doses of the composition containing Litsea cubeba extract for 30 days. Compared with the model control group, the percentage of blood glucose reduction in the high-dose group was significantly higher (P<0.05).

[0104] Table 9. Effects of the samples on fasting blood glucose in hyperglycemic model animals ( x ± s )

[0105] 2.4 Effects of compositions containing Litsea cubeba extract on glucose tolerance in hyperglycemic model animals See Tables 10 and 11. Mice were orally administered different doses of the composition containing Litsea cubeba extract for 30 days. There were no significant differences in blood glucose levels at 0.5 hours and 2 hours and the area under the blood glucose curve between the mice in each dose group and the model control group (P>0.05).

[0106] Table 10 Effects of the samples on glucose tolerance in hyperglycemic model animals ( x ± s )

[0107] Table 11 Effects of the samples on glucose tolerance in hyperglycemic model animals ( x ± s )

[0108] 3. Summary Under laboratory conditions, mice were orally administered the composition containing Litsea cubeba extract prepared according to this invention at doses of 0.5 g / kg·bw, 1.0 g / kg·bw, and 3.0 g / kg·bw for 30 days. In the 3.0 g / kg·bw dose group, the percentage decrease in blood glucose in hyperglycemic model mice was significantly higher than that in the control group (P<0.05). The composition had no significant effect on blood glucose levels at 0.5 hours and 2 hours after glucose administration, nor on the area under the blood glucose curve in hyperglycemic model mice (P>0.05). It also had no significant effect on fasting blood glucose in normal mice (P>0.05). This suggests that the submitted sample has a role in maintaining healthy blood glucose levels in animals.

[0109] Example 5 1. Materials and Methods 1.1 Sample The samples and placebo obtained in Example 1 were provided by China Medical Science, and both had essentially the same appearance and taste. The recommended oral dose for humans is one sachet twice daily.

[0110] 1.2 Subject Selection 1.2.1 Inclusion criteria: Select individuals with impaired glucose regulation (IGR) whose fasting blood glucose is 5.6-7 mmol / L (100-126 mg / dL) or whose 2-hour postprandial blood glucose is 7.8-11.1 mmol / L (140-200 mg / dL).

[0111] 1.2.2 Exclusion criteria: Diabetic patient.

[0112] Individuals under 18 years of age or over 65 years of age, pregnant or lactating women, or those allergic to the test sample.

[0113] Patients with complications in any of the following organs: heart, liver, kidney, etc., or those with other serious diseases, mental illness, or those taking glucocorticoids or other medications that affect blood sugar.

[0114] Those who cannot cooperate with dietary control, thus affecting the observation results.

[0115] Those with diabetic ketoacidosis, acidosis, or infection within the past 3 months.

[0116] The short-term use of items related to the test subject's function can affect the judgment of the results.

[0117] Those who do not meet the inclusion criteria, do not take the test sample as required, or whose data is incomplete and affects the observation results will not be included.

[0118] 1.3 Experimental Design and Grouping A between-group controlled design was employed, with participants grouped according to the requirements of randomized blinding. Participants were randomly assigned to either the trial group or the control group based on their glycated hemoglobin (HbA1c) or glycated serum protein (GSP) and blood glucose levels. Balance tests were conducted to account for key factors influencing the results, ensuring comparability between groups. Each group consisted of at least 50 participants. Prior to the trial, each participant was prescribed a diet according to their gender, age, activity level, ideal weight, and pre-existing lifestyle habits. This dietary control was maintained throughout the trial. Participants took the sample at the recommended daily dose for two consecutive months, which could be extended to four months if necessary.

[0119] 2. Observation Indicators 2.1 Security Indicators General physical signs include mental state, sleep, appetite, bowel movements, and blood pressure.

[0120] Routine blood, urine, and stool tests.

[0121] Liver and kidney function tests.

[0122] Chest X-ray, electrocardiogram, and abdominal ultrasound (only once before the test).

[0123] 2.2 Efficacy Indicators 2.2.1 Symptom Observation Observe the main symptoms such as excessive thirst, excessive hunger, fatigue, and frequent urination. Score the symptoms according to their severity. Calculate the scores before and after the trial. Observe the symptom improvement rate based on the improvement of the main symptoms (1 point for improvement is effective).

[0124] Table 12 Symptom Scoring Table

[0125] 2.2.2 Fasting blood glucose Observe the fasting blood glucose levels, the percentage decrease in fasting blood glucose, and the effectiveness rate of fasting blood glucose testing before and after the trial.

[0126] 2.2.3 Postprandial blood glucose 2 hours later Observe the blood glucose level 2 hours after consuming 100g of refined flour steamed buns before and after the trial, the percentage decrease in blood glucose 2 hours after the meal, and the effective rate of blood glucose 2 hours after the meal.

[0127] 2.2.4 Glycated hemoglobin or glycated serum protein Observe the changes in glycated hemoglobin or glycated serum protein before and after the food trial.

[0128] 2.2.5 Blood lipids Observe serum total cholesterol and serum triglyceride levels before and after the food trial.

[0129] 3. Data Processing and Result Determination For self-controlled data, paired t-tests can be used; for comparing means of two groups, independent t-tests are used. The latter requires a homogeneity of variance test. For non-normally distributed or unequally sized data, appropriate variable transformations should be performed until normality and homogeneity of variance are achieved, then the transformed data should be used for t-tests. If the transformed data still do not meet the requirements for normality and homogeneity of variance, a t' test or rank-sum test should be used instead. If the variances are homogeneous but the coefficient of variation is too large (e.g....),... CV For data with a percentage greater than 50%, the rank-sum test should be applied.

[0130] 3.1 Magnitude of Blood Glucose Decrease

[0131]

[0132] 3.2 The extent of decrease in glycated hemoglobin or glycated serum protein

[0133]

[0134] 3.3 Efficacy Judgment Criteria Effective: (1) Fasting blood glucose returns to normal (≤5.6 mmol / L) after the test, or the decrease in fasting blood glucose is ≥10%; (2) Blood glucose returns to normal 2 hours after the test (≤7.8 mmol / L), or the decrease in blood glucose 2 hours after the test is ≥10%.

[0135] Invalid: The valid standard has not been met.

[0136]

[0137]

[0138] 3.4 Indicator Determination 3.4.1 Fasting blood glucose ① A significant difference in the decrease of fasting blood glucose was observed before and after the experiment, with the average blood glucose returning to normal or decreasing by ≥10% after the experiment; ② The decrease in fasting blood glucose level or the magnitude of the decrease in fasting blood glucose was greater in the experimental group than in the control group after the experiment, with a significant difference; ③ The effective rate of fasting blood glucose reduction was higher in the experimental group than in the control group after the experiment, with a significant difference. If the above three conditions are met, the fasting blood glucose result of the test sample can be determined to be positive.

[0139] 3.4.2 Postprandial blood glucose 2 hours later ① A significant difference in the decrease of blood glucose 2 hours after a meal was observed between the test group and the control group after the test, with the average blood glucose returning to normal or decreasing by ≥10% after the test; ② A significant difference in the decrease of blood glucose 2 hours after a meal was observed in the test group or the magnitude of the decrease was greater than that in the control group; ③ A significant difference in the effective rate of blood glucose reduction 2 hours after a meal was observed in the test group than in the control group. Meeting these three conditions indicates a positive result for the blood glucose level 2 hours after a meal in the test sample.

[0140] 3.4.3 Glycated hemoglobin (or glycated serum protein) ① A significant difference in the decrease of glycated hemoglobin (or glycated serum protein) before and after the experiment; ② A significant difference in the decrease of glycated hemoglobin (or glycated serum protein) in the test group or a greater decrease in glycated hemoglobin (or glycated serum protein) than in the control group after the experiment. If both of these conditions are met, the glycated hemoglobin (or glycated serum protein) result of the test sample can be determined to be positive.

[0141] 3.4.4 Serum cholesterol ① The decrease in serum cholesterol was significantly different before and after the experiment; ② The decrease in serum cholesterol in the test group was significantly different from that in the control group after the experiment. If both of these conditions are met, the serum cholesterol result of the test sample can be considered positive.

[0142] 3.4.5 Serum triglycerides ① A significant difference in the decrease of serum triglycerides was observed before and after the experiment; ② A significant difference in the decrease of serum triglycerides in the test group compared to the control group after the experiment. If both of these conditions are met, the serum triglyceride level of the test sample can be considered positive.

[0143] 3.5 Result Determination The four indicators of fasting blood glucose, 2-hour postprandial blood glucose, glycated hemoglobin (or glycated serum protein), and blood lipids were not significantly elevated, and one of the two indicators of fasting blood glucose and 2-hour postprandial blood glucose was positive. Since there was no impact on the health of the group, it can be determined that the test sample has the effect of helping to maintain healthy blood glucose levels.

[0144] 4 Results The results of the double-blind observation were announced: those who took No. 2 received the composition containing Litsea cubeba disclosed in this invention, while those who took No. 1 received a placebo.

[0145] 4.1 Safety Observation 4.1.1 General Information: The initial experimental population consisted of 56 subjects in the control group and 56 subjects in the food trial group. No abnormalities were observed in the subjects' mental state, sleep, diet, or bowel and bladder function before and after the food trial. The control group had a male / female ratio of 24 / 32 and an average age of 46.55 ± 11.69 years. The food trial group had a male / female ratio of 24 / 32 and an average age of 45.98 ± 11.31 years.

[0146] 4.1.2 Abdominal ultrasound, electrocardiogram, and chest X-ray: No obvious abnormalities were found.

[0147] 4.1.3 Changes in weight, blood pressure, heart rate, urinalysis, stool routine examination, and blood routine examination indicators. No significant abnormalities were observed in weight, blood pressure, and heart rate in the trial group and control group before and after the food sample was tested. Urinalysis, stool analysis, and blood tests were all within the normal range. The results are shown in Table 13.

[0148] Table 13 Changes in body weight, blood pressure, heart rate, urinalysis, stool routine examination, and blood routine examination before and after the food trial ( )

[0149] 4.1.4 Changes in liver and kidney function indicators before and after the trial Before and after the food trial, as well as in the control group, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), albumin (ALB), globulin (GLOB), albumin / globulin ratio (A / G), total bilirubin (TBIL), direct bilirubin (DBIL), urea (BUN), creatinine (Cr), and uric acid (UA) were all within the normal range. The results are shown in Table 14.

[0150] Table 14 Changes in liver and kidney function indicators before and after the food trial ( )

[0151] 4.1.5 No obvious adverse reactions were observed during the trial period.

[0152] 4.2 Efficacy Observation 4.2.1 Symptom Observation After consuming the test sample for 60 days, the symptom scores of the test group were significantly different from those before the trial and those of the control group (p < 0.05). The improvement rate of clinical symptoms in the test group was significantly different from that in the control group (p < 0.05). The results are shown in Tables 15 and 16.

[0153] Table 15 Symptom Scores (Score Values) )

[0154] Table 16 Clinical symptom improvement rate (%)

[0155] 4.2.2 Fasting blood glucose Before the food trial, there was no significant difference in fasting blood glucose levels between the food trial group and the control group. P >0.05). There was no significant difference in fasting blood glucose levels after the trial in the control group compared to before the trial. P >0.05). The fasting blood glucose levels in the trial group after the trial were significantly different from their pre-trial levels and also significantly different from those in the control group after the trial. P <0.05), the decrease in fasting blood glucose and the effective rate of decrease in the experimental group were significantly different from those in the control group after the trial (the decrease was <0.05). P <0.05). Fasting blood glucose decreased by 10.15% after the trial. The results are shown in Table 17.

[0156] Table 17 Changes in fasting blood glucose before and after the food trial (mmol / L) )

[0157] Table 18. Efficacy rate of fasting blood glucose reduction before and after food trial (%)

[0158] 4.2.3 Postprandial 2-hour blood glucose Before the food trial, there was no significant difference in postprandial blood glucose levels between the food trial group and the control group (2 hours after the meal). P >0.05). In the control group, there was no significant difference in postprandial blood glucose levels 2 hours after the trial compared to before the trial. P >0.05). The 2-hour postprandial blood glucose levels in the trial group were significantly different from their pre-trial levels and also significantly different from those in the control group. P <0.05), the decrease in blood glucose level 2 hours after the meal in the trial group and the effective rate of decrease were significantly different from those in the control group. P <0.05). The blood glucose level in the trial group decreased by 9.18% 2 hours after the meal. The results are shown in Tables 19-20.

[0159] Table 19 Changes in blood glucose 2 hours after the meal before and after the food trial (mmol / L) )

[0160] Table 20. Efficacy rate of blood glucose reduction 2 hours after meal before and after food trial (%)

[0161] 4.2.4 Glycated hemoglobin Before the trial, there was no significant difference in glycated hemoglobin levels between the trial group and the control group. P >0.05). There was no significant difference in glycated hemoglobin levels in the control group after the trial compared to before the trial. P >0.05). There was no significant difference in glycated hemoglobin levels between the trial group and pre-trial levels, or between the trial group and the control group after the trial. P >0.05), after the trial, there was no significant difference in the decrease of glycated hemoglobin between the trial group and the control group. P >0.05). There was no significant difference in the percentage decrease in glycated hemoglobin after the trial group compared to the control group. P >0.05). The results are shown in Table 21.

[0162] Table 21 Changes in glycated hemoglobin before and after the food trial (%) )

[0163] 4.2.5 Serum cholesterol Before the trial, there was no significant difference in serum cholesterol levels between the trial group and the control group. P >0.05). There was no significant difference in serum cholesterol levels in the control group after the trial compared to before the trial. P>0.05). There were no significant differences in serum cholesterol levels between the trial group and pre-trial levels, or between the trial group and the control group after the trial. P >0.05). The results are shown in Table 22.

[0164] Table 22 Changes in serum cholesterol before and after the food trial (mmol / L) )

[0165] 4.2.6 Serum triglycerides Before the trial, there was no significant difference in serum triglycerides between the trial group and the control group. P >0.05). There was no significant difference in serum triglycerides in the control group after the trial compared to before the trial. P >0.05). There was no significant difference in serum triglycerides in the trial group after the trial compared to their pre-trial levels or to the control group after the trial. P >0.05). The results are shown in Table 23.

[0166] Table 23 Changes in serum triglycerides before and after the food trial (mmol / L) )

[0167] 4.2.7 Urinary glucose and urinary ketones There were no significant differences in urine glucose levels before and after the test, either within the same group or between groups. P >0.05), indicating that the sample had no significant effect on urinary glucose. No ketones were detected in urine before or after the food test. Results are shown in Table 24.

[0168] Table 24. Urine glucose and ketone body levels before and after the food trial (integrated values). )

[0169] 4.3 Dropout rate After a 60-day trial, 5 subjects in the control group were excluded due to intermittent use of the test product or inability to assess its effectiveness; 5 subjects in the experimental group were also excluded due to intermittent use of the test product or inability to assess its effectiveness. The final effective trial population consisted of 51 subjects in the control group and 51 subjects in the experimental group. The results are shown in Table 25.

[0170] Table 25 Test Dropout Rate

[0171] 5. Summary Using a self-control group and component-control method, voluntary subjects meeting the experimental conditions were selected and adhered to dietary control for 60 days while taking the test substance. Results showed that the composition containing Litsea cubeba disclosed in this invention improved symptoms of excessive thirst, polyphagia, fatigue, and polyuria, with a significant difference in the total improvement rate compared to the control group (P < 0.05). The fasting blood glucose levels in the test group before and after the test were significantly different from those in the control group after the test (P < 0.05). The 2-hour postprandial blood glucose levels in the test group before and after the test were also significantly different from those in the control group after the test (P < 0.05). The decrease in fasting blood glucose and 2-hour postprandial blood glucose levels and the effective rate of decrease in the test group were significantly different from those in the control group (P < 0.05). Fasting blood glucose decreased by 10.15% after the test, and 2-hour postprandial blood glucose decreased by 9.18% after the test. According to the "Test Method for Maintaining Healthy Blood Glucose Levels" in the "Methods for Functional Testing and Evaluation of Health Foods" (2023 Edition), the composition containing Litsea cubeba disclosed in this invention is suggested to have the effect of helping to maintain healthy blood glucose levels.

[0172] In summary, the composition containing Litsea cubeba extract disclosed in this invention is safe and has no toxic side effects, making it suitable for long-term use by individuals with metabolic syndrome.

[0173] Therefore, the composition disclosed in this invention uses Litsea cubeba extract, American ginseng extract, mulberry leaf extract, Astragalus membranaceus extract, and chromium-enriched yeast as main raw materials. Animal experiments have demonstrated that it helps maintain healthy blood glucose levels. Furthermore, network pharmacology and molecular docking have proven that the composition does not act through a single target, but rather embodies the synergistic effect of traditional Chinese medicine's "multi-component, multi-target, multi-pathway" approach. Its hypoglycemic mechanism uses PPARG as a key pivot, synergistically regulating the AGE-RAGE signaling pathway (targeting complications) and the endocrine resistance pathway (targeting core pathology) to achieve a comprehensive effect of improving insulin sensitivity, reducing metabolic inflammation and oxidative stress, and protecting target organs, thereby delaying the progression of diabetes and its complications.

[0174] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A composition comprising an extract of Litsea cubeba, characterized in that, The ingredients include the following parts by weight: 60-110 parts American ginseng extract, 80-130 parts mulberry leaf extract, 70-140 parts Litsea cubeba extract, 150-300 parts Astragalus membranaceus extract, and 2-15 parts chromium-enriched yeast.

2. The composition comprising Litsea cubeba extract according to claim 1, characterized in that, The ingredients include the following parts by weight: 70-105 parts American ginseng extract, 85-125 parts mulberry leaf extract, 80-130 parts Litsea cubeba extract, 165-250 parts Astragalus membranaceus extract, and 4-10 parts chromium-enriched yeast.

3. The composition comprising Litsea cubeba extract according to claim 1, characterized in that, It also includes excipients, which include one or more of soluble starch, maltodextrin, and dextrin.

4. The composition comprising Litsea cubeba extract according to claim 1, characterized in that, The American ginseng extract, mulberry leaf extract, Litsea cubeba extract, and Astragalus membranaceus extract are all water extracts.

5. The method for preparing the composition comprising Litsea cubeba extract as described in any one of claims 1-4, characterized in that, Weigh out Litsea cubeba extract, American ginseng extract, mulberry leaf extract, and astragalus extract in a certain proportion and mix them evenly. Then, take chromium-enriched yeast and mix it evenly with the mixed extract in an equal increment. Then, add the excipients, premix for 30 minutes, and mix evenly to obtain the composition.

6. The preparation method according to claim 5, characterized in that, Also includes: (1) Add 85% edible alcohol to the composition to make soft material, the amount of which is about 25% to 30% of the composition mass, and granulate it through a 14-mesh sieve to obtain wet granules; (2) Take the wet granules and dry them at 50℃~60℃, controlling the moisture content to about 5%, and granulate them through a 14-mesh sieve to obtain dry granules.

7. The use of the composition comprising Litsea cubeba extract as described in any one of claims 1-4, characterized in that, Its application in the preparation of medicines and health products for the prevention and improvement of diabetic complications.

Images