A green tea extract composition for reducing liver cell iron overload

Abstract

This invention discloses a green tea extract composition for reducing iron overload in hepatocytes, relating to the field of biomedical technology. The composition comprises a green tea extract as its active ingredient, rich in epigallocatechin-3-gallate, the content of which is identified by reversed-phase high-performance liquid chromatography and confirmed by comparison with the retention time of standard catechins. This composition, using natural green tea extract as its active ingredient, possesses both iron chelating and antioxidant effects. It can be used alone or in combination with clinical iron chelating agents to effectively reduce intracellular iron concentration, ferritin levels, and reactive oxygen species content in hepatocytes. It exhibits high safety and overcomes the side effects and limitations of existing synthetic chelating agents, providing a highly effective and safe adjunctive therapy for the treatment of β-thalassemia-related iron overload.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a green tea extract composition for reducing iron overload in hepatocytes. Background Technology

[0002] Beta-thalassemia is a common inherited hemoglobinopathic disorder characterized by ineffective erythropoiesis and chronic anemia. Severe cases require lifelong blood transfusions to sustain life. However, the human body lacks a physiological mechanism for actively excreting excess iron, and long-term blood transfusions can lead to systemic iron overload. Excess iron is converted into non-transferrin-bound iron (NTBI) and unstable plasma iron (LPI) through increased plasma transferrin saturation. These components are easily absorbed by parenchymal cells in organs such as the liver, making the liver a major target of iron toxicity.

[0003] Excess iron in cells catalyzes the generation of reactive oxygen species (ROS) through the Fenton reaction, causing widespread oxidative damage to lipids, proteins, and DNA. This is a key pathogenic mechanism leading to progressive liver damage, fibrosis, and even organ failure in patients. Currently, commonly used drugs for treating iron overload are synthetic iron chelators, including deferoxamine (DFO), deferoxone (DFP), and deferasirox (DFX). While these drugs can bind excess iron and promote its excretion, they have significant limitations: DFO requires intravenous administration, resulting in poor patient compliance and a high risk of side effects such as diarrhea, fever, and hearing loss; DFP may cause joint pain, arthropathy, and gastrointestinal reactions; and DFX carries the risk of kidney damage or gastrointestinal adverse reactions. Therefore, developing natural adjuvant therapies that combine iron chelating and antioxidant activity, have high safety profiles, and can synergize with existing chelators has become an urgent clinical challenge.

[0004] Green tea (Camellia sinensis) extract (GTE) is rich in polyphenolic compounds, among which epigallocatechin-3-gallate (EGCG) is the most active component, possessing both iron-chelating and potent antioxidant properties. Animal studies have confirmed that GTE and EGCG can reduce tissue iron deposition and lipid peroxidation. However, in physiologically relevant hepatocyte models, systematic comparisons and synergistic effects of GTE with clinical chelating agents are still incomplete, and the molecular mechanisms by which it regulates cellular iron metabolism require further elucidation. Summary of the Invention

[0005] The purpose of this invention is to provide a green tea extract composition for reducing hepatocyte iron overload, in order to solve the problem of hepatocyte iron overload and oxidative damage caused by blood transfusions for β-thalassemia, and to overcome the problems of existing synthetic iron chelating agents having side effects and being unable to simultaneously address iron clearance and oxidative damage.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a green tea extract composition for reducing iron overload in hepatocytes, wherein the active ingredient of the composition is green tea extract, the green tea extract is rich in epigallocatechin-3-gallate, and the content of epigallocatechin-3-gallate is identified by reversed-phase high-performance liquid chromatography and confirmed by comparison with the retention time of standard catechins.

[0007] Furthermore, the preparation method of the green tea extract includes the following steps:

[0008] S1. Fresh tea leaves are picked from the tea tree, and polyphenol oxidase is immediately inactivated in a microwave oven. Then, the tea leaves are ground into powder using a mixer.

[0009] S2. Add tea powder to deionized water at a ratio of 5 g / 100 ml, stir and extract at 80°C for 20 minutes to obtain the extract;

[0010] S3. The extract is filtered and then dried using a freeze dryer to obtain green tea extract.

[0011] Furthermore, the composition also contains at least one iron chelating agent, including deferoxamine, deferoxone, or derafloxacin.

[0012] Furthermore, the composition is used to reduce intracellular iron concentration in hepatocytes, and the green tea extract is used at a concentration of 1-30 μM, based on epigallocatechin-3-gallate equivalent, to reduce intracellular iron content in a dose-dependent manner.

[0013] Furthermore, the composition is used to reduce ferritin levels in hepatocytes, and the concentration of the green tea extract used is 10-30 μM, based on epigallocatechin-3-gallate equivalent, to reduce ferritin levels.

[0014] Furthermore, the composition has antioxidant activity, which can reduce the level of reactive oxygen species in iron-overloaded hepatocytes, and the green tea extract, at a concentration of 1-30 μM based on epigallocatechin-3-gallate equivalent, reduces the level of reactive oxygen species in a dose-dependent manner.

[0015] Furthermore, when the composition is used in combination with the iron chelating agent, the concentration of the green tea extract used in combination is 1-10 μM, calculated as epigallocatechin-3-gallate equivalent, and the concentration of deferoxamine or deferoxone used in combination is 10 μM. The combination of the two has a synergistic effect on reducing intracellular iron concentration and ferritin levels in hepatocytes.

[0016] Furthermore, when the composition is used in combination with deferoxone, it has a significant synergistic effect on reducing the level of reactive oxygen species in iron-overloaded hepatocytes, and the synergistic reduction effect is better than using the green tea extract alone or using deferoxone alone.

[0017] Furthermore, the composition works by targeting one or more genes related to iron metabolism and oxidative stress, including HAMP, TFRC, SOD1, SOD12, STAT1, MAPK14, APP, PTGS1, VCP, or TERT.

[0018] It regulates iron metabolism and oxidative stress response by interacting with the target site; and the epigallocatechin-3-gallate can form a stable binding with hepcidin with a binding energy of -9 kcal / mol. The binding sites include Pro7, His8, Ser9, Gln39, Arg40, Ser42, Gly42, and Lys106, which are stably bound through hydrogen bonds and hydrophobic interactions.

[0019] According to a second aspect of this disclosure, the use of the green tea extract composition of the first aspect in the preparation of an adjuvant remedy for treating or preventing hepatocyte iron overload and oxidative damage in β-thalassemia is also proposed.

[0020] Compared with existing technologies, this invention provides a green tea extract composition for reducing iron overload in hepatocytes. This composition uses natural green tea extract as the active ingredient, exhibiting high safety. Cell viability experiments show no significant toxicity to Huh7 hepatocytes within a concentration range of 1-30 μM (low LDH leakage rate). It overcomes the side effects of existing synthetic chelating agents and possesses both iron chelation and antioxidant effects, simultaneously addressing the two core issues of iron overload and oxidative damage. Used alone, it can dose-dependently reduce intracellular iron concentration, ferritin levels, and ROS content. Furthermore, at the same concentration, its ROS scavenging effect is superior to DFP. When used in combination with commonly used clinical chelating agents DFO and DFP, this compound exhibits a significant synergistic effect, enhancing iron mobilization and oxidative stress inhibition. The ROS scavenging effect is particularly optimal when combined with DFP, providing a new approach to optimizing existing iron overload treatment regimens. Furthermore, the overall preparation process is simple and cost-effective, employing water extraction and freeze-drying processes to effectively retain active ingredients such as EGCG. Reversed-phase high-performance liquid chromatography allows for precise control of the active ingredient content, ensuring product quality stability. The overall technical solution has a clear mechanism of action, regulating iron metabolism and oxidative stress pathways through multiple targets, providing a solid theoretical basis for its clinical application. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0022] Figure 1 The present invention provides HPLC-DAD analysis and cell viability detection chromatograms of green tea extract; wherein, Figure 1 a is the HPLC-DAD chromatogram of GTE; Figure 1 b shows the LDH leakage in Huh7 cells after treatment with DFO, DFP, and DFX (30 μM); Figure 1 c represents the LDH leakage of Huh7 cells after treatment with different concentrations of GTE (1-30 μM EGCG equivalent);

[0023] Figure 2 This invention provides graphs showing iron mobilization and ferritin levels in iron-overloaded Huh7 cells after different treatments; wherein... Figure 2 a represents the changes in intracellular iron concentration in the GTE, chelating agent alone, and combination therapy groups; Figure 2 b shows a comparison of cellular ferritin concentrations in each group;

[0024] Figure 3 This invention provides a graph showing the detection of intracellular ROS levels in iron-overloaded Huh7 cells; wherein, Figure 3 a represents the dose-dependent inhibition of ROS by a single chelating agent (1-30 μM); Figure 3 bd represents the synergistic inhibitory effect of GTE combined with different chelating agents on ROS; Figure 3 e represents a comparison of the ROS clearance effects of GTE and DFP alone and in combination;

[0025] Figure 4 The network analysis diagram of the interaction between GTE and iron overload provided by the present invention; wherein, Figure 4 a is an overlap diagram of the main components of GTE and genes related to iron overload; Figure 4 b represents the PPI network of overlapping genes; Figure 4 c is a bubble diagram of GO biological processes; Figure 4 d is a bubble diagram of GO molecule function; Figure 4 e is the molecular docking diagram of EGCG and hepcidin. Detailed Implementation

[0026] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0027] I. Preparation and Identification of Green Tea Extracts

[0028] 1. Raw materials and reagents

[0029] ① Raw materials: Freshly picked tender leaves and buds from tea trees in the tea garden, with withered leaves and impurities removed to ensure that the raw materials are free from mold and pests;

[0030] ②Reagents: Deionized water (DI), epigallocatechin-3-gallate (EGCG) standard (purity ≥98%), methanol (chromatographic grade), glacial acetic acid (analytical grade), 0.45μm filter membrane;

[0031] ③ Instruments: microwave oven, high-speed mixer, constant temperature water bath, magnetic stirrer, freeze dryer, high performance liquid chromatograph (equipped with diode array detector, HPLC-DAD), C18 column (4.6mm×250mm, 5μm);

[0032] 2. Preparation steps

[0033] S1. Raw material pretreatment: Immediately place the freshly picked tea leaves into a microwave oven and heat them on medium-high power for 3 minutes to quickly inactivate polyphenol oxidase (PPO) and prevent the oxidation and degradation of tea polyphenol active ingredients; then transfer the inactivated tea leaves to a high-speed blender and grind them for 5 minutes to obtain a uniform and fine tea powder for later use.

[0034] S2. Extraction process: Accurately weigh 5.0g of tea powder, add 100mL of deionized water, place in an 80℃ constant temperature water bath, start the magnetic stirrer (stirring speed 300r / min), and extract at a constant temperature for 20 minutes to ensure that the polyphenols in the green tea extract (GTE) are fully dissolved.

[0035] S3. Filtration and Drying: After extraction, the extract was filtered under reduced pressure through a 0.45μm filter membrane to remove solid impurities such as tea residue, and the clear filtrate was collected. The filtrate was transferred to a freeze dryer and the freeze drying conditions were set as follows: temperature -50℃, vacuum degree 0.1MPa, and continuous drying for 24 hours to obtain a light yellow loose powder of green tea extract (GTE). After sealing, it was stored in a cool and dry place.

[0036] S4. Identification and Quantification of Active Ingredients: Epigallocatechin-3-gallate (EGCG) in green tea extract was identified and quantified using reversed-phase high-performance liquid chromatography (HPLC-DAD). Specific chromatographic conditions are as follows:

[0037] Mobile phase: methanol-water-glacial acetic acid (volume ratio 40:60:1);

[0038] Detection wavelength: 280nm;

[0039] Flow rate: 1.0 mL / min;

[0040] Column temperature: 30℃;

[0041] Injection volume: 20 μL;

[0042] Identification method: GTE sample solution and EGCG standard solution were injected and analyzed separately. The presence of EGCG in GTE was confirmed by comparing the retention times of the two. The content of EGCG in GTE was calculated by using the standard curve method (with the concentration of EGCG standard as the abscissa and the peak area as the ordinate) to ensure that the EGCG content in the finished product is not less than 50% of the total polyphenols.

[0043] 3. Preparation Results

[0044] The final obtained green tea extract (GTE) was a pale yellow powder, easily soluble in water. HPLC-DAD analysis showed characteristic peaks in its chromatogram consistent with those of the EGCG standard (e.g., Figure 1 As shown in a), EGCG was confirmed as the core active ingredient, and its content met the requirements for subsequent experiments and applications.

[0045] II. Cellular Experiment Verification

[0046] 1. Cells and Culture Medium

[0047] ① Cell line: Human liver cancer cells (Huh7), purchased from a reputable cell bank, ensuring cell viability ≥95%;

[0048] ② Culture medium: RPMI 1640 medium, with 10% (v / v) heat-inactivated fetal bovine serum (FBS), 0.02% penicillin and 0.5% gentamicin added, stored in a refrigerator at 4°C, and brought to room temperature before use;

[0049] ③ Reagents and Instruments: Deferroamine (DFO), Deferrophenone (DFP), Deferraxis (DFX) (purity ≥98%), PBS buffer, LDH cytotoxicity colorimetric assay kit, ferriazine colorimetric method for iron content assay kit, ferritin ELISA kit, 2′,7′-dichlorofluorescein (DCFH-DA), fluorescence spectrometer, microplate reader, CO2 incubator, clean bench, centrifuge, 24-well cell culture plate.

[0050] 2. Cell Culture and Iron Loading Induction

[0051] ① Cell resuscitation and passage: Huh7 cells were removed from the liquid nitrogen tank and quickly placed in a 37°C water bath to thaw. After thawing, the cells were centrifuged (1000 r / min, 5 minutes) to remove the cryopreservation solution. The cells were resuspended in fresh culture medium, transferred to culture flasks, and cultured in a humidified incubator at 37°C and 5% CO2. When the cell confluence reached 80%-90%, the cells were passaged.

[0052] ② Cell seeding: Adjust the concentration of passaged Huh7 cells to 2×10⁻⁶. 5 Add 0.5 mL of RPMI 1640 medium containing antibiotics and 10% FBS to each well, seed in a 24-well culture plate, and incubate in an incubator for 24 hours until the cell confluence reaches 80%.

[0053] ③ Iron overload induction: Remove the old culture medium from the 24-well plate, add fresh RPMI 1640 medium containing antibiotics and 10% FBS, incubate for 10 hours, then replace with the same culture medium and continue incubation for another 10 hours (total 20 hours) to induce iron overload in cells via FBS; after induction, wash the cells twice with PBS buffer to remove unabsorbed iron ions and set aside.

[0054] 3. Experimental grouping and treatment

[0055] ① Control group: Add culture medium containing PBS buffer and incubate for 10 hours;

[0056] ② Individual chelating agent groups: Add medium containing 10 μM DFO, 10 μM DFP, and 10 μM DFX respectively, and incubate for 10 hours; Separate concentration gradient groups (1 μM, 30 μM DFO / DFP / DFX) are set up for the detection of reactive oxygen species (ROS) levels;

[0057] ③ GTE group alone: ​​Add medium containing 1 μM, 3 μM, 10 μM, and 30 μM GCG equivalent GTE respectively, and incubate for 10 hours;

[0058] ④ Combined drug treatment group: Add culture medium containing 10μM DFO+1μM / 3μM / 10μM GTE and 10μM DFP+1μM / 3μM / 10μM GTE respectively, and incubate for 10 hours; the GTE concentration is calculated as EGCG equivalent, and all groups are set up with 3 parallel replicates.

[0059] 4. Detection Indicators and Methods

[0060] ① Cell viability assay (LDH method):

[0061] After incubation, collect the cell culture supernatant from each well and measure the absorbance at 490 nm using an ELISA reader, following the instructions of the LDH cytotoxicity colorimetric assay kit.

[0062] Cell viability calculation formula: Cell viability (%) = (Experimental group absorbance value - Blank control group absorbance value) / (Normal control group absorbance value - Blank control group absorbance value) × 100%;

[0063] At a 20% inhibitory concentration (IC50) 20 To assess the cytotoxicity of GTE and its chelating agents.

[0064] ② Determination of intracellular iron content (ferroazine colorimetric method):

[0065] Discard the culture supernatant, add 200 μL of 200 mM sodium hydroxide solution to each well, and incubate overnight at 4°C to lyse the cells;

[0066] The next day, the cell lysis buffer was transferred to a centrifuge tube, centrifuged at 12,000 r / min for 10 minutes, and the supernatant was collected.

[0067] Following the instructions of the ferroazine colorimetric assay kit, ferroazine reagent was added to the supernatant and incubated at room temperature for 15 minutes. The absorbance was then measured at 562 nm using a microplate reader, and the intracellular iron concentration was calculated based on the iron standard curve.

[0068] ③ Ferritin level measurement (ELISA method):

[0069] The cell lysis process is the same as the "Intracellular Iron Content Determination" procedure, and the cell lysate supernatant is collected;

[0070] Following the instructions for the ferritin ELISA kit, add the standard, sample, and test reagent in sequence, measure the absorbance at 450 nm using an ELISA reader, and calculate the ferritin content based on the standard curve.

[0071] ④ Intracellular ROS level measurement (DCFH-DA fluorescence method):

[0072] After incubation, the supernatant was aspirated, and 10 μM DCFH-DA solution was added to each well. The wells were then placed in a 37°C, 5% CO2 incubator and labeled in the dark for 30 minutes.

[0073] After labeling, wash the cells three times with PBS buffer to remove unbound DCFH-DA;

[0074] Fresh culture medium was added, and fluorescence intensity (FI) was measured using a fluorescence spectrometer at an excitation wavelength (λex) of 493 nm and an emission wavelength (λem) of 522 nm. The fluorescence intensity value directly reflects the intracellular ROS level.

[0075] ⑤ Network pharmacology and molecular docking validation:

[0076] Active ingredient screening: The main components in GTE (epigallocatechin EGC, catechin C, epicatechin EC, epicatechin gallate ECG, EGCG) were analyzed using the TCMSP database to screen for active ingredients with oral bioavailability (OB) ≥ 30%.

[0077] Target prediction: Obtain the SMILES codes of the screened active ingredients from PubChem, predict the target of action using SwissTargetPrediction, and screen targets with a probability value > 0.

[0078] Disease target search: Using "iron overload" as the keyword, relevant genes were searched from the GeneCards and OMIM databases, and duplicates were removed after merging.

[0079] Overlapping target analysis: A Venn diagram was drawn using Venny to identify the overlapping portions (19 in total) between the GTE active ingredient target and the iron overload disease target.

[0080] PPI network construction: Overlapping target points were uploaded to the STRING database to obtain protein-protein interaction relationships, which were then imported into Cytoscape software for visualization analysis.

[0081] GO and KEGG enrichment analysis: Metascape was used to perform GO biological process (BP) and molecular function (MF) enrichment analysis on overlapping targets, and bubble diagrams were generated using Microbioinformatics;

[0082] Molecular docking: Molecular docking simulation was performed between EGCG, the core active ingredient of GTE, and hepcidin (PDB number: 3H0T), a hormone that regulates iron. The binding energy was calculated using AutoDock software, and the binding sites and interaction types were analyzed.

[0083] III. Experimental Results and Analysis

[0084] 1. Cell viability results

[0085] ①For example Figure 1 As shown in b, after treating Huh7 cells with 30 μM concentrations of deferoxamine (DFO), deferoxone (DFP), and deferasirox (DFX), the LDH leakage rate did not increase significantly, and the cell viability remained above 75%, indicating that the three chelating agents had no obvious cytotoxicity at this concentration.

[0086] ②For example Figure 1As shown in c, after treatment with 1-30 μM EGCG equivalent GTE, the LDH leakage rate of Huh7 cells remained below 20%, the cell viability was ≥80%, and no concentration-dependent toxicity was observed, demonstrating that GTE in this concentration range is safe for hepatocytes.

[0087] 2. Results of iron mobilization and ferritin levels

[0088] ① Iron mobilization effect: As shown in Figure 2a, GTE treatment alone reduced intracellular iron concentration in iron-overloaded Huh7 cells in a dose-dependent manner. The iron concentration in the 10 μM GTE group was significantly lower than that in the PBS control group (p<0.05). The use of 10 μM DFO, DFP, and DFX alone could effectively reduce intracellular iron levels. However, when GTE was combined with 10 μM DFO or 10 μM DFP, the reduction in iron concentration was significantly greater than that in the single treatment group (p<0.05), showing a significant synergistic iron mobilization effect.

[0089] ② Ferritin Levels: As shown in Figure 2b, treatment with 10 μM and 30 μM EGCG equivalent GTE reduced cellular ferritin levels by more than 70% compared to the PBS control group (p<0.0001); treatment with 10 μM, 30 μM DFO, DFP, and DFX alone also significantly reduced ferritin expression (p<0.01); when GTE was combined with 10 μM DFO or 10 μM DFP, ferritin levels were further reduced compared to the chelating agent groups alone (p<0.05), showing a significant synergistic effect; however, when GTE was combined with 10 μM DFX, there was no statistically significant difference in ferritin levels compared to the DFX-only treatment group (p>0.05).

[0090] 3. Results of ROS level regulation

[0091] ①Effects of using medication alone: ​​such as Figure 3 As shown in Figure a, treatment with 1-30 μM DFO, DFP, and DFX alone significantly reduced intracellular ROS levels in a dose-dependent manner (p<0.05); Figure 3 As shown in e, the ROS scavenging effect of 10 μM EGCG equivalent GTE alone was significantly better than that of 10 μM DFP (p<0.01), demonstrating that GTE has stronger direct antioxidant activity;

[0092] ② Effect of combined drug therapy: As shown in Figures 3b-d, when 10 μM DFO / DFP / DFX were combined with 1 μM and 10 μM GTE, the ROS level was significantly lower than that of the single drug groups (p<0.001), showing a synergistic antioxidant effect; among them, the ROS level of the 10 μM DFP and 10 μM GTE combination group decreased the most significantly, which was better than other combination groups and single drug groups (p<0.0001).

[0093] 4. Results of network pharmacology and molecular docking

[0094] ① Overlapping target analysis: such as Figure 4 As shown in Figure a, the Venn diagram shows that among the 45 differentially expressed genes regulated by GTE active ingredients and 1522 differentially expressed genes related to iron overload, there are 19 overlapping genes, which are the core targets of GTE in regulating iron overload.

[0095] ② PPI network: such as Figure 4 As shown in b, in the PPI network constructed from 19 overlapping genes, there are 17 closely connected hub proteins. The core cluster (purple nodes) includes iron metabolism-related genes HAMP (iron homeostasis regulator), TFRC (cellular iron uptake mediator), and oxidative stress-related genes SOD1 / 2 (ROS detoxification enzyme). The GTE direct regulatory cluster (green nodes) includes STAT1 (inflammatory stress signal hub), MAPK14 (stress apoptosis kinase), and APP (metal ion binding protein). The two types of clusters interact closely.

[0096] ③GO enrichment analysis: such as Figure 4 As shown in c, the GO biological process (BP) enrichment results indicate that overlapping genes are significantly enriched in "positive regulation of reactive oxygen species metabolism" (enrichment score -log60, p<-5), "monatomic cation homeostasis," and "heavy metal stress response," etc. Figure 4 As shown in d, molecular functions (MF) are enriched in "oxidoreductase activity", "transcriptional coactivator binding", and "protein phosphatase binding", which are closely related to iron metabolism and oxidative stress regulation.

[0097] ④ Molecular docking: such as Figure 4 As shown in e, the molecular docking results of EGCG and hepcidin show that the binding energy is -9 kcal / mol (lower than -7 kcal / mol, indicating stable binding). EGCG binds to the hydrophobic pocket of hepcidin and achieves stable binding through hydrogen bonds and hydrophobic interactions with key residues such as Pro7, His8, Ser9, Gln39, Arg40, Ser42, Gly42, and Lys106, providing molecular mechanism support for EGCG to regulate iron metabolism.

[0098] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A green tea extract composition for reducing iron overload in hepatocytes, characterized in that, The active ingredient of the composition is green tea extract, which is rich in epigallocatechin-3-gallate. The content of epigallocatechin-3-gallate is identified by reversed-phase high-performance liquid chromatography and confirmed by comparison with the retention time of standard catechins.

2. The green tea extract composition for reducing iron overload in hepatocytes according to claim 1, characterized in that, The preparation method of the green tea extract includes the following steps: S1. Fresh tea leaves are picked from the tea tree, and polyphenol oxidase is immediately inactivated in a microwave oven. Then, the tea leaves are ground into powder using a mixer. S2. Add tea powder to deionized water at a ratio of 5 g / 100 ml, stir and extract at 80°C for 20 minutes to obtain the extract; S3. The extract is filtered and then dried using a freeze dryer to obtain green tea extract.

3. The green tea extract composition for reducing iron overload in hepatocytes according to claim 1, characterized in that, The composition further comprises at least one iron chelating agent, including deferoxamine, deferoxone, or derarosi.

4. The green tea extract composition for reducing iron overload in hepatocytes according to claim 1, characterized in that, The composition is used to reduce intracellular iron concentration in hepatocytes, and the green tea extract is used at a concentration of 1-30 μM based on epigallocatechin-3-gallate equivalent, reducing intracellular iron content in a dose-dependent manner.

5. A green tea extract composition for reducing iron overload in hepatocytes according to claim 1, characterized in that, The composition is used to reduce ferritin levels in hepatocytes, and the concentration of the green tea extract used is 10-30 μM, based on epigallocatechin-3-gallate equivalent, to reduce ferritin levels.

6. The green tea extract composition for reducing iron overload in hepatocytes according to claim 1, characterized in that, The composition has antioxidant activity, can reduce the level of reactive oxygen species in iron-overloaded hepatocytes, and the green tea extract, at a concentration of 1-30 μM based on epigallocatechin-3-gallate equivalent, reduces the level of reactive oxygen species in a dose-dependent manner.

7. The green tea extract composition for reducing iron overload in hepatocytes according to claim 3, characterized in that, When the composition is used in combination with the iron chelating agent, the concentration of the green tea extract used in combination is 1-10 μM, calculated as epigallocatechin-3-gallate equivalent, and the concentration of deferoxamine or deferoxone used in combination is 10 μM. The combination of the two has a synergistic effect on reducing intracellular iron concentration and ferritin levels in hepatocytes.

8. A green tea extract composition for reducing iron overload in hepatocytes according to claim 7, characterized in that, When the composition is used in combination with deferoxone, it has a significant synergistic effect on reducing the level of reactive oxygen species in iron-overloaded hepatocytes, and the synergistic reduction effect is better than that of using the green tea extract alone or using deferoxone alone.

9. A green tea extract composition for reducing iron overload in hepatocytes according to claim 1, characterized in that, The composition works by targeting one or more genes related to iron metabolism and oxidative stress, including HAMP, TFRC, SOD1, SOD12, STAT1, MAPK14, APP, PTGS1, VCP, or TERT. It regulates iron metabolism and oxidative stress response by interacting with the target site; and the epigallocatechin-3-gallate can form a stable binding with hepcidin with a binding energy of -9 kcal / mol. The binding sites include Pro7, His8, Ser9, Gln39, Arg40, Ser42, Gly42, and Lys106, which are stably bound through hydrogen bonds and hydrophobic interactions.

10. The use of a green tea extract composition as described in any one of claims 1-9 in the preparation of an adjuvant remedy for treating or preventing hepatocyte iron overload and oxidative damage in β-thalassemia.

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