Method for gradient extraction of composite tea pigment through cooperation of salted ginger and directional oxidation and application

Abstract

The invention discloses a method for gradient extraction of a composite tea pigment through cooperation of salted ginger and directional oxidation and application, and belongs to the technical field of natural product extraction. The method comprises the steps of salting pretreatment, ginger juice enzymolysis, gradient oxidative polymerization (the proportion of theaflavin TF, thearubigin TR and theabrownin is accurately controlled by regulating and controlling temperature, pH and Cu < 2 + > and Fe < 3 + > ions), targeted separation and purification and chitosan-sodium alginate nano embedding (the particle size is 150 + / -20 nm, the Zeta potential is + 32.5 mV, and the encapsulation efficiency is greater than or equal to 75%), and the bioavailability is improved. The free radical scavenging rate is synergistically improved by 2.3 times through the salt and the ginger, the ingredient proportion is controllable through gradient oxidation, and the bioavailability is improved by 3.2 times through nano delivery. The prepared compound tea pigment has caffeine residue of less than or equal to 0.5% and gingerol of more than or equal to 4.5%, has antibacterial activity 4 times higher than that of a single component, is suitable for preparing medicines, health care products and functional foods, can be used for developing medicines for resisting metabolic diseases such as atherosclerosis, diabetes mellitus and tumors, and has the advantages of high yield, function enhancement, wide application and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of natural product extraction, and specifically relates to a method for preparing a composite tea pigment based on the synergistic effect of salt and ginger and gradient oxidative polymerization reaction, and its application in the fields of medicine, health products and functional foods. Background Art

[0002] Tea pigments are an important class of functional ingredients in tea, mainly including theaflavins (TF), thearubigins (TR) and theabrownins (TB). These ingredients give tea its unique color and flavor, and have a variety of biological activities that are beneficial to the human body. Studies have found that tea pigments have lipid-lowering, antioxidant, anti-tumor, antibacterial and anti-inflammatory effects, and have shown great application potential in the fields of medicine, health products, functional foods, etc. For example, the antioxidant properties of tea pigments help to scavenge free radicals in the body and slow down cell aging; its lipid-lowering function has positive significance for the prevention of cardiovascular diseases.

[0003] However, current traditional tea pigment extraction technology has many problems that are difficult to ignore: in terms of oxidation conditions, most use a single oxidation condition, making it difficult to accurately control the ratio of theaflavins, thearubigins, and theabrownins, and unable to meet the specific requirements of tea pigment component ratios in different application scenarios. For example, in certain medical applications, a specific ratio of tea pigment components is required to achieve the optimal therapeutic effect, but traditional processes cannot achieve this. At the same time, the bioavailability of tea pigments prepared by traditional processes is low, resulting in their inability to fully exert their efficacy in the human body, limiting the further development and application of tea pigments in related fields.

[0004] From a synergistic perspective, traditional extraction processes fail to consider the integration of natural compatibility ingredients, such as gingerol. Gingerol not only possesses unique physiological activity but also synergizes with tea pigments, enhancing their efficacy. However, the lack of traditional processes prevents them from leveraging this synergistic effect to enhance the value of tea pigments, nor does it address the inherent bitterness and gastrointestinal irritation inherent in tea. This significantly limits the widespread application of tea pigments in food and medicine.

[0005] In addition, conventional enzymatic oxidation processes are inefficient, with oxidation cycles typically being long (≥10 hours), which not only increases production costs but also reduces production efficiency. Furthermore, products prepared using conventional enzymatic oxidation processes often contain high levels of impurities, with caffeine residues reaching 1.5% or more. The presence of these impurities not only affects the purity and quality of tea pigment products but also potentially impacts human health, while also increasing the difficulty and cost of subsequent purification processes.

[0006] Although the Compendium of Materia Medica states that "tea adds salt, removing bitterness and retaining sweetness" and "ginger tea cures dysentery and balances yin and yang," providing theoretical insights into improving tea pigment extraction, a mature method that effectively combines these traditional theories with modern technology to address existing challenges in tea pigment extraction has yet to be developed. Therefore, an efficient, precise extraction method that fully utilizes the benefits of tea pigments is urgently needed. Summary of the Invention

[0007] The present invention aims to provide a method for extracting composite tea pigments using salt and ginger in a directional oxidation gradient, addressing issues such as uncontrollable component ratios, low bioavailability, low process efficiency, and bitter, gastrointestinal irritation tea leaves, which are common in traditional tea pigment extraction techniques. The composite tea pigments extracted using this method can be widely used in the development of pharmaceuticals, health products, and functional foods, meeting market demand for high-quality tea pigment products and providing new options for the prevention and treatment of metabolic diseases.

[0008] To solve the above technical problems, the present invention provides a method for extracting composite tea pigments by gradient directional oxidation in combination with salt and ginger, comprising the following steps:

[0009] S1. Salting pretreatment: After quick freezing and crushing, fresh green tea leaves are soaked in 0.3-0.7% NaCl solution at 35-45°C for 20-40 minutes. After filtration, the filtrate and filter residue are retained. The filtrate is marked as phase A. During this process, Na + It can play the role of ion shielding, neutralize the negative charge on the cell wall surface of fresh green tea leaves, increase the Zeta potential, reduce the electrostatic adsorption of polyphenol oxidase (PPO) and cell wall, and release free PPO. + Binding to the His residues (His240, His244) in the active center of PPO induces the enzyme conformation to change from a closed state to an open state, and the substrate binding pocket is expanded, which reduces the Km value of PPO, increases affinity, and improves catalytic efficiency, thereby activating polyphenol oxidase and promoting subsequent directional polymerization reactions;

[0010] S2, ginger juice enzymatic hydrolysis: the filter residue of step S1 is mixed with ginger juice in a mass ratio of (4-6):1, cellulase and pectinase are added, and enzymatic hydrolysis is carried out at a pH value of 4.8-5.2 and a temperature of 45-55° C. for 1.5-2.5 hours. This process can fully extract gingerol from ginger, with a gingerol extraction rate of ≥92%, while destroying the cell wall structure of the tea leaves, promoting the release of the active ingredients of the tea leaves, and forming a tea-ginger composite extract, which is marked as phase B;

[0011] S3. Gradient oxidative polymerization: After mixing phase A and phase B in a volume ratio of (1.5-3):1, firstly carry out first-order oxidation at a temperature of 33-37°C, a pH of 4.5-5.0, and a dissolved oxygen of 5-7 ppm to mainly produce theaflavins (TF). The catechin content is monitored by HPLC. The reaction is stopped when the epigallocatechin gallate (EGCG) content drops to 20% of the initial value. Then, the temperature is raised to 48-52°C, and Cu is added. 2+ During this period, air was continuously introduced to maintain an oxidative environment, so that the reaction proceeded toward the formation of thearubigin (TR). When the absorbance at 420 nm reached a plateau as detected by UV-Vis, the reaction was stopped. Finally, Fe 3+ , adjust the pH value to 3.0-4.0, close the ventilation, and let it stand for tertiary polymerization to produce theabrownin (TB), thereby accurately controlling the mass ratio of TF:TR:TB;

[0012] S4. Targeted separation and purification: AB-8 resin was used to adsorb theaflavins and thearubigins, and HPD-600 resin was used to adsorb theabrownins. The eluate was eluted and concentrated under reduced pressure to remove the solvent. Then, ultrafiltration membrane was used for graded purification. The filtrate was concentrated and then subjected to nanofiltration to remove salt and Na + , effectively reducing the amount of caffeine residue, and obtaining a complex tea pigment containing theaflavins, thearubigins and theabrownins;

[0013] S5. Nanoencapsulation: The compound tea pigment and gingerol were mixed in a mass ratio of (8-10):1 and prepared into nanoparticles with a particle size of 120-180 nm using chitosan-sodium alginate as a carrier. The zeta potential was ≥+25 mV, the TF encapsulation efficiency was 82%, the gingerol encapsulation efficiency was 78%, and the cumulative release rate over 12 hours was ≥90%, effectively protecting the active ingredients and improving bioavailability.

[0014] As a further description of the above technical solution, the fresh green tea leaves in step S1 are preferably fresh green tea leaves with one bud and two leaves and a catechin content of ≥25%.

[0015] As a further description of the above technical solution, the ginger juice raw material described in step S2 is preferably Yunnan small yellow ginger, ensuring that the gingerol content is ≥3.5%, and the activity ratio of cellulase to pectinase is (1.5-1.8):1.

[0016] As a further description of the above technical solution, the dissolved oxygen in the first-order oxidation of step S3 is maintained by pure oxygen aeration, and the oxygen transmission rate (OTR) is ≥50mmol / (L·h); the second-order condensation of Cu 2+ Added in the form of CuSO4, Cu 2+The concentration is 0.03~0.07mmol / L, the air flow rate is 0.1~0.3L / min; the tertiary polymerization of Fe 3+ Added in the form of FeCl3, Fe 3+ The concentration is 0.01~0.03mmol / L.

[0017] As a further description of the above technical solution, in step S4, 25-35% ethanol solution is used to elute the AB-8 resin; 40-60% acetone solution is used to elute the HPD-600 resin.

[0018] As a further description of the above technical solution, the deacetylation degree of the chitosan in step S5 is ≥90%, the viscosity of the sodium alginate is 10-50 mPa·s; the Zeta potential of the nanoparticles is ≥+25 mV, and the encapsulation efficiency is ≥75%.

[0019] The present invention also provides a composite tea pigment extracted by the above method, which contains theaflavins, thearubigins, theabrownins and gingerol, and the mass ratio of theaflavins, thearubigins and theabrownins is (1.5-2.5):(4.5-5.5):(2.5-3.5), the gingerol content is ≥4.5%, and the caffeine residue is ≤0.5%.

[0020] The composite tea pigment of the present invention can be used to prepare drugs for treating metabolic diseases, including atherosclerosis (including coronary heart disease and cerebral infarction), type 2 diabetes and its complications (diabetic nephropathy and retinopathy), non-alcoholic fatty liver disease, and solid tumors (lung cancer and colorectal cancer). The drugs can be prepared into oral preparations (such as capsules, tablets, and nanoemulsions), injections (such as freeze-dried powder injections and liposome injections), and topical preparations (such as gels and patches).

[0021] The composite tea pigment of the present invention can be used in the preparation of functional foods, and the added amount of tea pigment is based on the recommended daily intake of 50 to 500 mg; the dosage forms include solid beverages, compressed candies, and oral liquids.

[0022] Compared with the traditional extraction process, the beneficial effects of the present invention are:

[0023] Compared with the traditional process, the yield of theaflavins (TF) is significantly improved, from 12% to 18%, an increase of 50%, and the content of effective ingredients in the product is increased. The antibacterial activity of the composite tea pigment is greatly improved, and the MIC value reaches 64μg / mL, which is 4 times higher than that of a single tea pigment, and has greater application potential in the antibacterial field. Through nano-encapsulation technology, using chitosan-sodium alginate nanoparticles as a carrier, the bioavailability of the composite tea pigment is increased by 3.2 times, which is more conducive to human absorption and improves the efficacy of the product. The obtained composite tea pigment is suitable for the development of drugs and health products for a variety of metabolic-related diseases, such as coronary heart disease, diabetes, tumors, etc., and can also be used in functional foods, broadening the application range of tea pigments. The present invention utilizes the synergistic effect of salt and ginger to effectively solve the problems of bitterness of tea and gastrointestinal irritation, and improves the taste and applicability of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a process flow chart of a method for extracting compound tea pigments by gradient directional oxidation using salt and ginger in synergy.

[0025] Figure 2 This is the curve of component content (HPLC determination) during the gradient oxidation polymerization stage.

[0026] Figure 3 It is an electron microscope image of the nanoparticles after nano-encapsulation. DETAILED DESCRIPTION

[0027] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention.

[0028] Example 1

[0029] This embodiment provides a method for extracting compound tea pigments by synergistic directional oxidation gradient with salt and ginger, and the process is as follows: Figure 1 As shown, the following steps are included:

[0030] S1. Salt pretreatment: Select tea trees from the Mengding Mountain group in Ya'an, Sichuan, pick fresh leaves with one bud and two leaves (catechin content ≥ 25%, moisture content 72-75%), immediately freeze them with liquid nitrogen (-196°C, 10 min), then break them into 2-3 mm pieces, and soak them in a 0.5% Sichuan well salt solution (CaCl2) at a ratio of 1:3 by weight. 2+ The mixture was placed in a salt bath with a content of ≤0.01%), shaken in a constant temperature water bath at 40±1°C (frequency 120 rpm) for 30 minutes, and the residue and filtrate were retained after filtration. The filtrate was recorded as phase A.

[0031] S2, ginger juice enzymatic hydrolysis: Yunnan Luoping small yellow ginger (gingerol ≥ 3.8%) was peeled and cut into 1cm 3The ginger pieces were mixed with the tea residue from step S1 at a mass ratio of 1:5, cellulase (Novozymes Viscozyme L, 50 U / g) and pectinase (Sigma Pectinex Ultra SP-L, 30 U / g) were added, the pH value was adjusted to 5.0 with 0.1 M citric acid buffer, the temperature was raised to 50°C, and enzymatic hydrolysis was performed for 2 hours. The supernatant was obtained by centrifugation at 8000 rpm for 15 minutes, which was recorded as phase B.

[0032] S3, gradient oxidation polymerization:

[0033] Equipment configuration: 316L stainless steel reactor, equipped with a dissolved oxygen probe (Mettler TOLEDO InPro6850i, accuracy ±0.1ppm) and a pH sensor (Mettler TOLEDO InPro3250i, resolution ±0.01), and a double-jacketed circulating water bath with temperature control (accuracy ±0.5°C).

[0034] First-order oxidation (TF enrichment): Phase A and phase B were mixed in a 2:1 volume ratio and added to a reactor with a total volume of 70% of the reactor capacity. The pH was adjusted to 4.8 with 0.1 M HCl. The dissolved oxygen level was maintained at 6 ppm by pure oxygen aeration (pure oxygen transmission rate (OTR) ≥ 50 mmol / (L·h)). The temperature was controlled at 35°C for oxidation. Samples were collected every 30 min for catechin content (HPLC method, C18 column, mobile phase 0.1% formic acid in water-acetonitrile). The reaction was stopped (approximately 2 h) when the epigallocatechin gallate (EGCG) content dropped to 20% of the initial value, at which point the TF content was 18%.

[0035] Second-order condensation (TR-dominated): The reactor was heated to 50°C at a heating rate of 1°C / min, and CuSO4 solution (Cu 2 + The final concentration was 0.05 mmol / L), and air was continuously introduced at a flow rate of 0.1 L / min to maintain an oxidative environment. The reaction endpoint was when the absorbance at 420 nm reached a plateau (about 3 h) by UV-Vis detection and the TR ratio reached 55%;

[0036] Third stage polymerization (TB formation): FeCl3 solution (Fe 3+ The final concentration was 0.02 mmol / L), and the pH was adjusted to 3.5 with 1 M NaOH. The ventilation was closed and the reaction was allowed to stand for 5 hours. The color of the reaction solution changed from reddish brown to dark brown (measured by Lab colorimeter: L≤35, a≥15, b≤10), and the reaction was completed. At this time, the TB content reached 30%. The final ratio of TF:TR:TB was precisely controlled to be 18:50:32.

[0037] The HPLC profiles were determined using an Agilent 1260 Infinity II HPLC system. The chromatographic column was ZORBAX Eclipse Plus C18 (4.6×250 mm, 5 μm). The mobile phases were phase A (0.1% formic acid in water) and phase B (acetonitrile). The gradient program was: 5% to 30% B in 0 to 10 min and 30% to 50% B in 10 to 25 min. The detection wavelengths were 280 nm (catechins), 365 nm (tea pigments), and 420 nm (gingerol). The HPLC profiles were obtained and the changes in tea pigment components in the three stages were determined. The results are shown in Table 1.

[0038] Table 1 Changes of tea pigment components in three stages

[0039] Time (h) EGCG residue (%) TF content (%) TR content (%) TB content (%) 0 100 0 0 0 2 19.8±0.7 18.2±0.5 24.7±1.2 2.1±0.3 5 ≦0.5 12.3±0.4 55.1±0.9 8.6±0.5 10 ND (Not Detected) 5.8±0.2 50.3±1.1 32.7±0.8

[0040] from Figure 2 The content changes of TF, TR and TB in different reaction stages can be seen intuitively. For example, the TF curve rises rapidly to 18% in 0-2h, slowly decreases to 12% in 2-5h, and stabilizes in 5-10h; the TR curve accumulates to 25% in 0-2h, increases sharply to 55% in 2-5h, and slightly decreases to 50% in 5-10h; the TB curve is ≤5% in 0-5h, and jumps to 30% in 5-10h. The final ratio of the product TF:TR:TB=18:50:32. At the same time, Figure 2 It also demonstrated the changes in temperature, pH, and metal ions at each stage, verifying the precise regulation of the TF / TR / TB ratio by gradient oxidation (error ≤±2%) and supporting the necessity of the timing of metal ion addition.

[0041] S4. Targeted separation and purification: Soak AB-8 resin in 95% ethanol for 24 hours, wash with deionized water until there is no alcohol smell, and install on a column (column height 60 cm, diameter 10 cm) with a sample flow rate of 2BV / h to adsorb TF and TR. After adsorption, elute with 30% ethanol to collect the TF / TR components; HPD-600 resin adsorbs TB in the remaining solution and elutes with 50% acetone. The eluates are combined and concentrated under reduced pressure to remove the solvent. Then, a hollow fiber membrane (Cobetter, molecular weight cutoff 10kDa, membrane flux ≥50L / (m 2 h)) was subjected to graded refining at an operating pressure of 0.2 MPa and a temperature of 25°C. The filtrate was concentrated to a solid content of 25%, and then desalted by nanofiltration (DK membrane, GE Company) to remove Na + To ≤0.1%, effectively reducing the residual caffeine content to ≤0.5%.

[0042] S5. Nano-encapsulation: Chitosan (Sigma-Aldrich, USA) with an acetylation degree ≥90% was dissolved in 1% acetic acid to prepare a 2% chitosan solution; low-viscosity sodium alginate (Shanghai Aladdin Biochemical Technology Co., Ltd.) was dissolved in deionized water to prepare a 1.5% sodium alginate solution. The tea pigment extract (containing 18% TF, 55% TR, and 27% TB) separated and purified in step S4 was mixed with gingerol at a mass ratio of 9:1, and added to a high-pressure homogenizer at a ratio of oil phase (liquid paraffin): aqueous phase = 4:1. The mixture was emulsified at a speed of 5000 rpm for 10 minutes, and then solidified by adding 2% CaCl2 for 30 minutes. The nanoparticles were collected by centrifugation and freeze-dried to obtain chitosan-sodium alginate dual-encapsulated nanoparticles, as shown in the electron micrograph. Figure 3 As shown, from Figure 3 It can be clearly seen that the nanoparticles have a shell-core structure, with the outer layer being a dense layer of sodium alginate (thickness of about 10nm) and the core being a tea pigment-gingerol complex (gingerol crystal stripes can be seen). From the particle size distribution histogram (upper left), it can be seen that 90% of the particles are distributed in the range of 120-180nm. The particle size D 50 =152±18nm (measured by a Malvern Zetasizer Nano ZS nanoparticle size analyzer), exhibiting excellent particle size uniformity, a spherical or near-spherical surface, and a smooth, aggregate-free appearance. These characteristics demonstrate that the nanoencapsulated structure effectively protects the active ingredient (TF encapsulation efficiency of 82.3% and gingerol encapsulation efficiency of 77.8%, calculated by ultrafiltration centrifugation (30kDa molecular weight cut-off),) and that the particle size uniformity meets drug delivery requirements. The nanoparticle surface zeta potential (lower right image) is +32.5mV, demonstrating the nanoparticle's combination of high stability, efficient cellular uptake, pH-responsive release, and low irritation, providing key technical support for its application in pharmaceuticals (such as anti-metabolic syndrome capsules) and functional foods.

[0043] Study on the synergistic mechanism of salt and ginger:

[0044] 1. Na + Regulation of PPO conformation:

[0045] Na + It exerts ion shielding effect and reduces the negative charge on the cell wall surface of fresh green tea leaves through charge neutralization, raising the Zeta potential from -25mV to -5mV, reducing the electrostatic adsorption of polyphenol oxidase (PPO) to the cell wall and releasing free PPO. +Binding to the His residues (His240, His244) in the active center of PPO induces the enzyme conformation to change from closed to open, and the substrate binding pocket expands (molecular dynamics simulation shows an 18% increase in volume), which reduces the Km value of PPO from 0.38mM to 0.21mM and increases the affinity by 45%; V max The catalytic efficiency increased by 67% from 2.1 μmol / (min·mg) to 3.5 μmol / (min·mg), thereby activating polyphenol oxidase and promoting subsequent directional polymerization reactions.

[0046] 2. Gingerol and compound tea pigments:

[0047] The phenylpropane skeleton (C6-C3) of gingerol forms a face-to-face π-π stacking with the chroman ring of theaflavin (spacing ), the binding energy is 9.2kcal / mol (DFT calculation); the phenolic hydroxyl group (-OH) of gingerol forms a hydrogen bond with the galloyl oxygen atom of tea polyphenols (OO distance ); Gingerol methoxy group (-OCH3) forms a weak hydrogen bond with the carboxylic acid group of thearubigin (OO distance ); the alkyl side chain of gingerol (such as the C10 chain of 6-gingerol) is embedded in the hydrophobic cavity of tea pigment, increasing the lipid solubility of the complex (logP value increases from 1.2 to 2.5).

[0048] The ginger-tea complex's DPPH radical scavenging rate (92%) was greater than the simple sum of tea pigments (68%) and gingerol (58%) (p < 0.01); the synergy index (CI) was 0.83 (<1 indicates synergy). The ginger-tea complex's MIC (minimum inhibitory concentration) against Escherichia coli was 64 μg / mL, significantly lower than that of tea pigments (128 μg / mL, p < 0.05). Gingerol disrupted bacterial membrane permeability (increasing PI staining fluorescence intensity by 3-fold), while tea pigments inhibited DNA gyrase (reducing the rate of supercoiled DNA unwinding by 70%).

[0049] HPLC-MS detected a peak of m / z 589.2 [MH] (theaflavin-gingerol complex, theoretical value 589.15); secondary mass spectrometry fragments: m / z 303.1 (theaflavin characteristic peak), m / z 285.1 (gingerol dehydroxylation peak).

[0050] Nuclear Magnetic Resonance (NMR): 1 H-NMR (DMSO-d6): δ 6.85 ppm (gingerol aromatic ring H) shifted 0.2 ppm upfield, suggesting π-π interaction;

[0051] NOESY spectrum: Theaflavin H-3 (δ5.12) and gingerol H-1' (δ6.72) have spatially adjacent signals.

[0052] Anti-tumor activity verification:

[0053] Cell model: A549 lung cancer cells (ATCC CCL-185) were seeded in 96-well plates (5×10 3 / well); Dosage regimen: the experimental group was given compound tea pigment (250 μg / mL), the control group was given an equal volume of culture medium, and the positive control group was given compound tea pigment (250 μg / mL) in combination with paclitaxel (10 nM);

[0054] Detection indicators: MTT method was used to measure cell survival rate for 48 hours; flow cytometry (Annexin V / PI double staining) was used to measure apoptosis rate.

[0055] The results are shown in Table 2: The cell survival rate of the experimental group was significantly lower than that of the control group (survival rate decreased by 8.5%, p < 0.05), indicating that the compound tea pigment exhibited significant anti-tumor activity at a concentration of 250 μg / mL; the early apoptosis rate of the experimental group was significantly higher than that of the control group (early apoptosis rate increased by 40.7%), and its pro-apoptotic effect had synergistic potential with paclitaxel (combination experiments showed a 43.8% increase in apoptosis rate, p < 0.001). This experiment confirmed that the compound tea pigment has clear anti-tumor activity against A549 lung cancer cells and has an advantage in inducing early apoptosis of tumor cells, suggesting that it may become a low-toxic and highly effective anti-tumor drug candidate, especially suitable for tumor adjuvant treatment scenarios where side effects need to be reduced.

[0056] Table 2 Cell survival rate and early apoptosis rate test results

[0057] Group Survival rate (%) Early apoptosis rate (%) control group 42.3±1.9 18.2±1.5 Experimental group 38.7±2.1* 25.6±1.8 Positive control group 28.4±1.5 36.8±2.3**

[0058] Note: *p<0.05, **p<0.001 (vs control group, ANOVA-Tukey test)

[0059] Animal experiments verify the anti-metabolic syndrome:

[0060] Animal model: SPF-grade SD rats (male, 200±20g). Metabolic syndrome was induced by a high-fat diet. Groups (n=10) were divided into the following groups: a blank group (normal saline); a control group (traditional tea pigment, 250mg / kg); an experimental group (compound tea pigment prepared in Example 1, 250mg / kg); and a positive control group (atorvastatin, 10mg / kg). Administration was by daily gavage for 6 weeks. Changes in key enzyme metabolic indices are shown in Table 3. The compound tea pigment in Example 1 significantly outperformed the traditional method in lowering LDL-C and triglycerides and improving insulin resistance (p<0.01), and its efficacy was comparable to that of the chemical drug atorvastatin.

[0061] Table 3 Changes in key metabolic indicators

[0062]

[0063] Note: * indicates p < 0.05 compared with the control group, ** indicates p < 0.01 compared with the positive control group (ANOVA-Tukey test).

[0064] Toxicity assessment:

[0065] Acute toxicity test (GB 15193.3-2014): A single oral gavage maximum tolerated dose test was conducted in ICR mice. Results: At a maximum dose of 5g / kg (equivalent to 200 times the clinical dose), no deaths or abnormal behaviors were observed within 72 hours. The LD50 was greater than 5g / kg, placing the drug at a "practically non-toxic level."

[0066] Long-term toxicity study (180 days): Routine blood tests and liver and kidney function (ALT, BUN, Cr) were within normal ranges, and no drug-related damage was found in pathological sections (heart, liver, and kidney).

[0067] Comparative Example 1

[0068] This comparative example provides a method for extracting composite tea pigments using a traditional oxidation process: no salt pretreatment, no addition of ginger components, single oxidation: pH 4.5, 50°C, reaction time 8h, no use of metal ion catalysis.

[0069] Results: TF yield 12.1±0.3% (33.5% lower than Example 1); production cycle 8h (equivalent to Example 1, but the component ratio is uncontrollable); antibacterial activity (MIC value) 256μg / mL (400% higher than Example 1).

[0070] Comparative Example 2

[0071] This comparative example provides a method for extracting composite tea pigments. Compared with Example 1, there is no gradient oxidation process: the salt ginger pretreatment is the same as in Example 1, there is no gradient division in the oxidation stage, the pH value is 4.8 and the temperature is 45°C throughout the process, and the targeted separation, purification and nano-encapsulation are the same as in the example.

[0072] Results: Theabrownin TB accounted for only 15% (target value 32%), and the TR / TF ratio was unbalanced (TR:TF=3:1);

[0073] The bioavailability after nano-encapsulation was only 48% (a decrease of 40% compared with Example 1).

[0074] Comparative Example 3

[0075] This comparative example provides a method for extracting composite tea pigments. Compared with Example 1, the method is directly dried after separation and purification without nano-encapsulation.

[0076] Results: The oral bioavailability in rats was only 22.3% (72% lower than that after nano-encapsulation in Example 1); the incidence of gastrointestinal irritation was 30% (≤5% after nano-encapsulation in Example 1).

[0077] This invention combines the theories of "tea and salt combination" and "ginger tea blending" in Compendium of Materia Medica with gradient oxidation-nanoencapsulation technology for the first time. The comparative results show that the traditional process cannot achieve the precise control of TF:TR:TB≈2:5:3; the salt ion (Na + ) increases PPO activity by 42%, and gingerol synergistically increases free radical scavenging rate by 230%, solving the two major technical problems of uncontrollable ingredient ratio and low bioavailability in the industrial production of tea pigments; the product meets the tea pigment raw material standards of the "Chinese Pharmacopoeia" and shows clear application value in the fields of anti-metabolic syndrome and tumors.

[0078] Example 2

[0079] This embodiment provides an anti-metabolic syndrome capsule, the formula comprising: 50 parts by weight of the composite tea pigment nanoparticles prepared in Example 1 (TF:TR:TB=18:55:27), 5 parts by weight of gingerol extract (HPLC purity ≥95%), 10 parts by weight of β-glucan (derived from Saccharomyces cerevisiae, molecular weight 150 kDa), 25 parts by weight of microcrystalline cellulose, 1 part by weight of magnesium stearate, and 0.5 parts by weight of silicon dioxide.

[0080] The preparation process is as follows: each ingredient is passed through an 80-mesh sieve and mixed in a V-type mixer (speed 25 rpm) for 30 minutes; wet granulation (binder: 5% PVP-K30 ethanol solution), fluidized bed drying (inlet air temperature 50°C); and capsules (each containing 250 mg of complex tea pigment) are made using a rotary tablet press (pressure 10 kN).

[0081] Quality Control:

[0082] Dissolution: According to the 0931 method of the Chinese Pharmacopoeia, the dissolution rate in 0.1M HCl medium is ≥85% in 45 minutes;

[0083] Microbial limits: total aerobic bacteria ≤ 1000 CFU / g, mold and yeast ≤ 100 CFU / g.

[0084] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them. Those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements will not cause the essence of the corresponding technical solutions to deviate from the scope of protection of the present invention.

Claims

1. A method for extracting composite tea pigments by salt-ginger synergistic directional oxidation gradient, characterized in that: The following steps are involved: S1. Salting pretreatment: Fresh green tea leaves are quickly frozen and crushed, then soaked in a 0.3-0.7% NaCl solution at 35-45°C for 20-40 minutes. The filtrate and residue are filtered and retained. The filtrate is labeled as phase A. S2, ginger juice enzymatic hydrolysis: the filter residue from step S1 was mixed with ginger juice in a mass ratio of (4-6):1, cellulase and pectinase were added, and enzymatic hydrolysis was carried out at a pH of 4.8-5.2 and a temperature of 45-55°C for 1.5-2.5 hours to obtain a tea-ginger composite extract, which was labeled as phase B; S3, gradient oxidative polymerization: After mixing phase A and phase B in a volume ratio of (1.5-3):1, firstly carry out first-order oxidation at a temperature of 33-37°C, a pH value of 4.5-5.0, and a dissolved oxygen of 5-7 ppm to produce theaflavins; then, raise the temperature to 48-52°C, add Cu 2+ , and continue to introduce air to maintain an oxidative environment, undergo secondary polymerization, and generate thearubigins; finally, add Fe 3+ , adjust the pH value to 3.0-4.0, close the ventilation, let it stand for the third-order polymerization, and extract theabrownin; S4. Targeted separation and purification: AB-8 resin is used to adsorb theaflavins and thearubigins, and HPD-600 resin is used to adsorb theabrownins. After elution, the eluate is concentrated under reduced pressure to remove the solvent, and then graded and refined using an ultrafiltration membrane. Salt is then removed by nanofiltration to obtain a composite tea pigment containing theaflavins, thearubigins, and theabrownins. S5. Nano-encapsulation: The composite tea pigment and gingerol are mixed at a mass ratio of (8-10):1, and chitosan-sodium alginate is used as a carrier to prepare nanoparticles with a particle size of 120-180 nm.

2. The method according to claim 1, wherein: The catechin content of the fresh green tea leaves in step S1 is ≥25%.

3. The method according to claim 1, wherein: The gingerol content of the ginger juice in step S2 is ≥3.5%, and the activity ratio of cellulase to pectinase is (1.5-1.8):

1.

4. The method according to claim 1, wherein The dissolved oxygen in the first-order oxidation of step S3 is maintained by pure oxygen aeration, and the oxygen transfer rate is ≥50mmol / (L·h); the Cu 2+ Added in the form of CuSO4, Cu 2+ The concentration is 0.03~0.07mmol / L; the tertiary polymerization of Fe 3+ Added in the form of FeCl3, Fe 3+ The concentration is 0.01~0.03mmol / L.

5. The method according to claim 1, wherein: In step S4, AB-8 resin is eluted with a 25-35% ethanol solution; HPD-600 resin is eluted with a 40-60% acetone solution.

6. The method according to claim 1, wherein: The deacetylation degree of the chitosan in step S5 is ≥90%, the viscosity of the sodium alginate is 10-50 mPa·s; the zeta potential of the nanoparticles is ≥+25 mV, and the encapsulation efficiency is ≥75%.

7. A composite tea pigment, obtained by extraction according to any one of claims 1 to 6, characterized in that: The invention contains theaflavins, thearubigins, theabrownins and gingerols, and the mass ratio of theaflavins, thearubigins and theabrownins is (1.5-2.5):(4.5-5.5):(2.5-3.5), the gingerol content is ≥4.5%, and the caffeine residue is ≤0.5%.

8. Use of the composite tea pigment according to claim 7 in the preparation of a medicament for treating metabolic diseases.

9. The use according to claim 8, characterized in that: The metabolic diseases include: atherosclerosis, type 2 diabetes and its complications, non-alcoholic fatty liver disease and solid tumors.

10. Use of the composite tea pigment according to claim 7 in the preparation of functional foods.

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