Application of theaflavin compounds in preparation of products for inhibiting DPP-4 (dipeptidyl peptidase-4)

Abstract

The invention belongs to the technical field of medicines, and particularly provides a novel application of a theaflavin compound in preparation of a product for inhibiting dipeptidyl peptidase-4 (DPP-4), and the theaflavin compound is one or more of theaflavin-3-gallate (TF2A), theaflavin-3 '-digallate (TF3) and theaflavin (TF). The research systematically proves that TF2A, TF3 and TF have new application of inhibiting DPP-4 for the first time: in-vitro enzymology and fluorescence experiments show that theaflavin can inhibit DPP-4 and interact with DPP-4, and molecular docking further shows possible binding configuration and key action sites of theaflavin. In vivo, the activity of DPP-4 in serum and different tissues of high-fat diet mice can be reduced, serum glucagon-like peptide-1 (GLP-1) can be up-regulated, and glycometabolism and inflammatory phenotype can be improved. Therefore, the theaflavin compound disclosed by the invention can be applied to preparation of products for inhibiting DPP-4, and has a good development prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to the use of a theaflavins compound in the preparation of a dipeptidyl peptidase-4 (DPP-4) inhibitor. BACKGROUND

[0002] Diabetes is a metabolic disease characterized by chronic hyperglycemia, mainly caused by insulin secretion and (or) defects [1] . At present, the prevalence of diabetes is increasing year by year, which is the main reason for the increase of morbidity and mortality in the world, especially in Asia, and has become an important global public health problem [2] . Type II diabetes is the main type of diabetes, accounting for 90% to 95% of the incidence of diabetes [3] , mainly manifested as insulin resistance and (or) damage to pancreatic beta cells. Diabetes not only damages multiple organs in the body, but also causes a variety of complications, seriously affecting human physical health and quality of life [4] , so the prevention and treatment of diabetes has become increasingly important.

[0003] At present, the drugs for type II diabetes mainly include sulfonylureas, thiazolidinediones, GLP-1 (glucagon-like peptide-1) receptor agonists, sodium-glucose co-transporter 2 inhibitors and dipeptidyl peptidase-4 (DPP-4) inhibitors [5] . Among them, DPP-4 inhibitors have received widespread attention due to their low incidence of hypoglycemia, no weight gain and edema, good cardiovascular safety and other characteristics, and have become a popular target for the treatment of type II diabetes [6] . DPP-4 inhibitors inhibit DPP-4, thereby inhibiting the inactivation of GLP-1 and glucose-dependent insulinotropic polypeptide (GIP), increasing the levels of endogenous GLP-1 and GIP, and promoting the release of insulin from pancreatic beta cells to improve hyperglycemia [7] . Inhibition of DPP-4 can prevent 80% to 97% of GLP-1 from being degraded and prolong its half-life to 2 to 40 hours [8] . In addition, DPP-4 inhibitors can also improve the function of pancreatic beta cells, promote wound healing, and reduce the risk of bone fracture [9]However, many DPP-4 inhibitors have many side effects, so it is the focus and continuous efforts to find a new, economic, safe DPP-4 inhibitor. In fact, in addition to drug control, many diabetes cases can be prevented by changing lifestyle, one of which is a healthy diet. A large number of studies have shown that daily consumption of cereals, fruits and vegetables, fish, eggs, milk and other foods have a positive effect on blood glucose regulation, especially natural products such as polyphenols, flavonoids, alkaloids and other foods have been found to have a strong inhibitory effect on DPP-4

[10] Compared with drugs, foods and their bioactive ingredients have many advantages, such as low toxicity and side effects, multiple physiological functions, multiple pathways, multiple targets, natural safety, high compliance, etc.

[0004] Theaflavins (TFs) are dimeric polyphenols formed by enzymatic oxidation and condensation of catechins during the fermentation of fresh tea leaves. They are important components that determine the color, aroma, and flavor of black tea and its functions. The main monomers include theaflavin (TF), theaflavin-3-gallate (TF2A), theaflavin-3'-gallate (TF2B), and theaflavin-3,3'-digallate (TF3). As key substances that determine the color and taste of black tea, theaflavins are widely present in black tea, oolong tea, and their extracts, and have attracted attention in the development of tea beverages and functional foods. Theaflavins have been reported to have various biological activities, such as antioxidant, anti-inflammatory, antibacterial, antitumor, and antiviral activities. Based on these excellent biological activities, theaflavins are considered to have high development potential. However, there is no report on the role of theaflavins in inhibiting DPP-4.

[0005] REFERENCES

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[0007] [2] Zimmet P Z, Magliano D J, Herman W H, et al. Diabetes: a 21st century challenge [J]. The lancet Diabetes & endocrinology, 2014, 2(1): 56-64.

[0008] [3] Kahn S E, Cooper M E, Del Prato S. Pathophysiology and treatment of type 2 diabetes: perspectives on the past, present, and future [J]. The Lancet, 2014, 383(9922): 1068-1083.

[0009] [4] Ben-Avraham S, Harman-Boehm I, Schwarzfuchs D, et al. Dietary strategies for patients with type 2 diabetes in the era of multi-approaches; review and results from the Dietary Intervention Randomized Controlled Trial (DIRECT) [J]. Diabetes research and clinical practice, 2009, 86: S41-S48.

[0010] [5] Gor D, Lee T A, Schumock G T, et al. Adherence and persistence with DPP-4 inhibitors versus pioglitazone in type 2 diabetes patients with chronic kidney disease: a retrospective claims database analysis [J]. Journal of Managed Care & Specialty Pharmacy, 2020, 26(1): 67-75.

[0011] [6]Howse P M, Chibrikova L N, Twells L K, et al. Safety and efficacy of incretin-based therapies in patients with type 2 diabetes mellitus and CKD: a systematic review and meta-analysis [J]. American Journal of Kidney Diseases, 2016, 68(5): 733-742.

[0012] [7]Omar B, Ahrén B. Pleiotropic mechanisms for the glucose-lowering action of DPP-4 inhibitors [J]. Diabetes, 2014, 63(7): 2196-2202.

[0013] [8]Drucker D J. The role of gut hormones in glucose homeostasis [J]. The Journal of clinical investigation, 2007, 117(1): 24-32.

[0014] [9]Zhu Hai. New oral hypoglycemic drugs: DPP-4 inhibitors [J]. Jiangsu Health Care, 2022(03): 14-15.

[0015]

[10] Zheng Y, Ley S H, Hu F B. Global aetiology and epidemiology of type 2 diabetes mellitus and its complications [J]. Nature reviews endocrinology, 2018, 14(2): 88-98. SUMMARY

[0016] In view of the deficiencies of the prior art, the purpose of the present application is to propose the use of theaflavins in inhibiting DPP-4.

[0017] The second purpose of the present application is to propose a DPP-4 inhibitor, and the effective component is theaflavins.

[0018] The technical solutions for achieving the above purposes of the present application are as follows:

[0019] The use of theaflavins in inhibiting DPP-4, wherein the theaflavins are selected from one or more of theaflavin-3-gallate (TF2A), theaflavin-3,3'-bisgallate (TF3), and theaflavins (TF).

[0020] In vitro enzymatic and fluorescence experiments showed that the theaflavins could inhibit DPP-4 and interact with it.

[0021] Theaflavin compounds were further shown to have possible binding configurations and key interaction sites with DPP-4 through molecular docking.

[0022] The theaflavins can reduce DPP-4 activity in serum and some tissues and upregulate serum GLP-1 levels in vivo.

[0023] Preferably, the theaflavins can improve glucose metabolism and relieve inflammation.

[0024] This invention proposes the use of the theaflavins in the preparation of foods, health products or drugs that inhibit DPP-4.

[0025] Furthermore, the food, health product, or drug may also contain food- or pharmaceutically acceptable excipients.

[0026] The present invention further proposes a DPP-4 inhibitor, the active ingredient of which is a theaflavins, wherein the theaflavins are selected from one or more of theaflavins-3-gallate (TF2A), theaflavins-3,3'-bisgallate (TF3) and theaflavins (TF).

[0027] The dosage form of the DPP-4 inhibitor can be tablets, pills, liquids, powders, chewable tablets, or soft capsules.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] To address the research gaps in the field of DPP-4 inhibition by theaflavins TF2A, TF3, and TF, this invention provides the first systematic evaluation and verification of the in vitro inhibitory activity and molecular-level binding characteristics of these theaflavins against DPP-4, including in vitro enzymatic (IC50) assays. 50 Experimental and fluorescence experiments showed that theaflavins can inhibit DPP-4 and interact with it. Molecular docking further revealed its possible binding configuration and key action sites. In vivo, it reduced the activity of DPP-4 in serum and different tissues of mice on a high-fat diet, upregulated serum glucagon-like peptide-1 (GLP-1), and improved glucose metabolism and inflammatory phenotype.

[0030] This invention not only expands the bioactivity spectrum of theaflavins, but also provides new applications for theaflavins in the preparation of related health foods and pharmaceuticals with DPP-4 inhibition functions. Attached Figure Description

[0031] Figure 1 ICs for TF, TF3 and TF2A 50 curve.

[0032] Figure 2 The fluorescence emission spectra of DPP-4 under the action of TF, TF3 and TF2A are shown.

[0033] Figure 3 This is a two-dimensional interaction diagram of molecular docking between TF, TF3, and TF2A and DPP-4.

[0034] Figure 4 This study investigated the effects of intervention with theaflavins (TF, TF3, and TF2A) on glucose tolerance, fasting blood glucose, insulin, DPP-4 activity, and GLP-1 levels in mice fed a high-fat diet. * indicates a significant difference compared to the control group, and # indicates a significant difference compared to the model group.

[0035] Figure 5 The effects of intervention with theaflavins (TF, TF3, and TF2A) on the levels of inflammatory cytokines IL-6 and TNF-α in mice fed a high-fat diet. * indicates a significant difference compared to the control group, and # indicates a significant difference compared to the model group.

[0036] Figure 6 The effects of theaflavins (TF, TF3, and TF2A) intervention on DPP-4 activity in different tissues. * indicates a significant difference compared to the control group, and # indicates a significant difference compared to the model group. Detailed Implementation

[0037] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0038] Unless otherwise specified, all test materials and instruments used in this instruction manual are commercially available.

[0039] In this specific embodiment, TF2A was purchased from Chengdu Efa Biotechnology Co., Ltd., and TF3 and TF were purchased from Chengdu Purifa Technology Development Co., Ltd. Unless otherwise specified, the operations and materials used in the embodiments are all known in the art.

[0040] Example 1: IC 50 Measurement

[0041] The IC of the three substances studied in this invention 50The simplified procedure for the assay is as follows: 25 μL of sample was pre-incubated with 25 μL of glycyl-prolyl-p-nitroaniline hydrochloride (GP-pNA) (3.2 mM) at 37 °C for 10 min. Then, 50 μL of DPP-4 (0.02 U / mL) (Sigma-Aldrich, St. Louis, MO, USA) was added. After reacting for 60 min, the reaction was terminated by adding 100 μL of 1 M sodium acetate solution. The absorbance was measured at 405 nm using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). The DPP-4 inhibition rate (DI) was calculated using the following formula. A DI–log C curve was plotted, and the concentration at which the inhibition rate reached 50% was calculated from the curve as the IC50. 50 .

[0042]

[0043] A(sample): Absorbance when the substrate, sample, and enzyme are present simultaneously.

[0044] A (sample blank): Absorbance when substrate and sample coexist in the absence of enzyme.

[0045] A (positive): Absorbance of the enzyme and substrate in the absence of a sample.

[0046] A (negative): Absorbance in the absence of enzymes and sample.

[0047] See results Figure 1 The IC50 values ​​of the three compounds for DPP-4 50 The corresponding values ​​are TF = 1453 μM, TF3 = 2417 μM, and TF2A = 628 μM, respectively. Figure 1 As shown in the figure. Within the concentration range tested in this study, TF2A exhibited stronger inhibitory activity, while TF and TF3 showed relatively weaker inhibitory activity.

[0048] Example 2: Fluorescence Spectroscopy Measurement

[0049] The fluorescence spectra of DPP-4 with and without TF, TF3, and TF2A were measured using a fluorescence spectrophotometer at an excitation wavelength of 280 nm. Emission spectra were recorded in the scanning wavelength range of 290–500 nm, with both the excitation and emission slit widths adjusted to 5 nm and a scan rate of 600 nm / min. Before measurement, DPP-4 (0.1 mg / mL) and the analyte solution (0.8 mg / mL) were mixed at a volume ratio of 1:1 and allowed to equilibrate at room temperature for 10 min before measurement.

[0050] Because DPP-4 contains tryptophan and tyrosine residues, it exhibits strong intrinsic fluorescence. When the excitation wavelength is set to 280 nm, DPP-4 shows a strong emission peak around 330 nm. With the gradual addition of TF2A, TF3, and TF to the system, the intensity of the characteristic peak of DPP-4 gradually decreases, as shown below... Figure 2 As shown, the three compounds interacted with DPP-4 and caused the quenching of the protein's intrinsic fluorescence.

[0051] Example 3: Molecular docking

[0052] The DPP-4 protein (PDB:5J3J) was selected and processed using the Protein Preparation Workflow: hydrogen atoms and partial charge were added, missing side chains / loops were reconstructed, and the energy was minimized to RMSD under an OPLS force field. Ligands were prepared using the LigPrep module. Hydrogen atoms were added to the ligand molecules to correct the bond order, and all ionization states of the ligands were determined using Epik at pH 7.0 ± 2. OPLS force fields were employed to generate up to 32 possible conformations for each ligand at a given pH. Furthermore, the ligands were desalted as needed to generate tautomers, and the prepared ligands were used for docking studies. The grid files were generated by the Receptor Grid Generation module, with a grid around the ligand. Inner box and one Outer box. Receptor and ligand-based meshes were generated using the XP docking protocol for docking. This process was accomplished using the GLIDE module. During docking, conformational poses were added internally and these poses were screened. In the initial stage, the ligand was centered and then allowed to rotate around a rotatable angle. Unfavorable binding modes were removed based on docking scores and geometric filters. Subsequently, mesh-based force field evaluation and docking refinement were performed using the OPLS-AA force field. The docking process involves predicting the conformation and orientation of the ligand within the target molecule binding site. Finally, the optimal conformation was selected based on the docking score for subsequent analysis.

[0053] Molecular docking showed that the docking fractions of the three substances with DPP-4 were: TF -10.65 kcal / mol, TF2A -8.67 kcal / mol, and TF3 -4.88 kcal / mol. Two-dimensional interaction diagram ( Figure 3 The results show that TF and TF2A can occupy the active cavity well and form multi-point hydrogen bonds to contact the aromatic / hydrophobic groups; TF3 is larger in size, with some substituents exposed to the solvent, resulting in relatively weaker pairing within the cavity. Overall, all three compounds can enter the active site of DPP-4 and interact with it.

[0054] Example 4: Animal Experiment

[0055] Forty-eight male C57 BL / 6J mice (6 weeks old) were selected and housed in a specific pathogen-free animal facility. After one week of acclimatization, the mice were randomly divided into six groups (n=8 per group): (1) Normal group: fed a low-fat diet and administered a carrier solution by gavage; (2) Model group: fed a high-fat diet and administered a carrier solution by gavage; (3) Sit group: fed a high-fat diet and administered 10 mg / kg sitagliptin by gavage; (4) TF group: fed a high-fat diet and administered 100 mg / kg TF solution by gavage (i.e., 100 mg per kg of mouse body weight per day); (5) TF2A group: fed a high-fat diet and administered 100 mg / kg TF2A solution by gavage; (6) TF3 group: fed a high-fat diet and administered 100 mg / kg TF3 solution by gavage. The intervention lasted for eight weeks. At week 8, mice were sacrificed after fasting for 12 hours overnight and separated at 4°C (3500 rpm, 15 min) to obtain serum, and tissues such as intestines and pancreas were collected.

[0056] In week 7, after a 12-hour overnight fast, all mice underwent an oral glucose tolerance test by being given an oral glucose solution (2 g / kg body weight). Blood glucose levels were measured using a blood glucose meter (Contour TS1816; Bayer, Leverkusen, Germany) at 0, 15, 30, 60, 90, and 120 min, collected from the tail vein.

[0057] like Figure 4 As shown in (a), (b), and (c), the model group exhibited significantly higher glucose tolerance, fasting blood glucose, and insulin levels compared to the control group, indicating successful model establishment and glucose metabolism disorder. After intervention, sitagliptin (Sit), TF, TF3, and TF2A all showed varying degrees of improvement. Specifically, AUC was significantly lower in all intervention groups compared to the model group; regarding fasting blood glucose, Sit, TF3, and TF2A were significantly lower than in the model group, with TF showing a decreasing trend; regarding insulin, the model group showed a significant increase, while TF3, TF2A, and Sit were significantly lower than in the model group, suggesting a certain alleviating effect on insulin resistance or β-cell burden.

[0058] Example 5: Assay of serum glucose, insulin, GLP-1 and DPP-4 activities

[0059] Mice obtained from the animal experiments in Example 4 were tested. Serum glucose was measured using a commercial kit from Nanjing Jiancheng Bioengineering Institute, according to the manufacturer's instructions; serum insulin and GLP-1 were measured using an enzyme immunoassay kit, according to the manufacturer's instructions; serum DPP-4 activity was measured as per the IC50 values ​​in Example 1. 50 The determination method.

[0060] Serum DPP-4 activity and GLP-1 levels:

[0061] Compared with the blank group, the model group showed increased DPP-4 activity. Figure 4 (d) and GLP-1 levels decreased ( Figure 4 (e) indicates an imbalance in the incretin axis. After intervention, Sit and the three theaflavins (TF, TF3, TF2A) significantly reduced serum DPP-4 activity, while GLP-1 levels rebounded. These changes are consistent with the action chain of "inhibiting DPP-4 → reducing GLP-1 degradation → increasing endogenous GLP-1", and are also consistent with the improvement direction of the aforementioned glucose metabolism outcome indicators.

[0062] Example 6: Determination of serum TNF-α and IL-6 levels

[0063] Mice obtained from the animal experiments in Example 4 were tested. Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were measured using an enzyme immunoassay kit.

[0064] like Figure 5 As shown, serum IL-6 and TNF-α levels were significantly higher in the model group compared to the control group, indicating the activation of systemic inflammation. Intervention with the three theaflavins (TF, TF3, and TF2A) significantly reduced IL-6 and TNF-α levels compared to the model group. Combined with the aforementioned results of decreased DPP-4 activity and increased GLP-1, these changes are consistent with the mechanism of action of "inhibiting DPP-4 → reducing incretin degradation → relieving inflammation," indicating that theaflavins have a certain alleviating effect on high-lipid-induced inflammatory responses.

[0065] Example 7: DPP-4 activity assay in different tissues

[0066] Mice obtained from the animal experiments in Example 4 were tested. Using GP-pNA as a chromogenic substrate, 10 μl of serum and tissue samples were mixed with 190 μl of 2 mM GP-pNA in 96-well plates. DPP-4 activity was kinetically determined by measuring the rate of pNA release from the chromogenic substrate (405 nm) over 10 minutes at 37°C.

[0067] See Figure 6A high-fat diet significantly upregulated hepatic DPP-4 activity; Sit, TF2A, and TF3 significantly inhibited this upregulation, while no statistically significant difference was observed in the TF group. In the ileum, a high-fat diet also increased DPP-4 activity; Sit, TF2A, TF3, and TF all significantly reversed this change. In the jejunum, there were no significant differences in DPP-4 activity among the groups. In the pancreas, DPP-4 activity in the model group was significantly higher than in the control group, and Sit, TF2A, TF3, and TF were all significantly lower than in the model group. Overall, the changes in DPP-4 activity induced by a high-fat diet were tissue-specific, with the TF2A group showing a more pronounced downward trend in DPP-4 activity, suggesting a better inhibitory effect.

[0068] discuss:

[0069] This study focuses on the novel application of theaflavins in inhibiting DPP-4, and establishes a mutually supportive chain of evidence from in vitro enzyme inhibition experiments, fluorescence quenching, molecular docking, and in vivo verification in mice. 50 The results showed that the potency ranking was TF2A (628 μM) > TF (1453 μM) > TF3 (2417 μM). Fluorescence spectroscopy results indicated that the above compounds could interact with DPP-4 and cause quenching of the protein's intrinsic fluorescence. Molecular docking results further revealed their possible binding configuration and key action sites with DPP-4. In vivo, the three compounds reduced serum and different tissue DPP-4 activity, upregulated serum GLP-1 levels, improved glucose tolerance and fasting blood glucose, and decreased inflammatory factors to varying degrees in a high-fat diet model. These results suggest that TF2A, TF3, and TF may improve glucose metabolism and alleviate inflammation by inhibiting DPP-4 activity and reducing GLP-1 degradation.

[0070] Although the present invention has been described above through embodiments, those skilled in the art should understand that any improvements and modifications made to the present invention without departing from its spirit and essence should fall within the protection scope of the present invention.

Claims

1. The use of theaflavins in inhibiting DPP-4, characterized in that, The theaflavins are selected from one or more of theaflavins-3-gallate, theaflavins-3,3'-bisgallate, and theaflavins.

2. The use of theaflavins in inhibiting DPP-4, characterized in that, The theaflavins can inhibit DPP-4 and interact with it.

3. The use of theaflavins in inhibiting DPP-4, characterized in that, The theaflavins were further demonstrated through molecular docking to reveal their possible binding configurations and key binding sites with DPP-4.

4. The use of theaflavins in inhibiting DPP-4, characterized in that, The theaflavins can reduce DPP-4 activity in serum and some tissues and upregulate serum GLP-1 levels in vivo.

5. The use of theaflavins in inhibiting DPP-4, characterized in that, The theaflavins can improve glucose metabolism and relieve inflammation.

6. Use of the theaflavins of claim 1 in the preparation of foods, health products or pharmaceuticals that inhibit DPP-4.

7. The use according to claim 5, characterized in that, The food, health product, or drug may also contain food- or pharmaceutically acceptable excipients.

8. A DPP-4 inhibitor, characterized in that, The active ingredient is a theaflavins, which are selected from one or more of theaflavins-3-gallate, theaflavins-3,3'-bisgallate, and theaflavins.

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