CN114468293B - Theaflavine nutrition preparation for enhancing skeletal muscle movement characteristics and application thereof - Google Patents

Abstract

The invention provides an extraction method of a theaflavin-containing black tea extract for enhancing skeletal muscle movement characteristics, a theaflavin nutrition mixture for enhancing skeletal muscle movement characteristics, and application of the theaflavin nutrition mixture in preparation of a nutritional supplement for enhancing skeletal muscle movement characteristics. The theaflavin nutrition mixture can effectively inhibit the expression of IL-6, IL-1, TNF-alpha of inflammatory factors in skeletal myositis reaction, has positive protection effect on the change of muscle mechanical characteristics under the action of inflammation, and is a formula consisting of natural product extracts, and has the characteristics of natural products.

Description

Theaflavine nutrition preparation for enhancing skeletal muscle movement characteristics and application thereof

Technical Field

The invention relates to a theaflavine nutrition preparation with the functions of regulating skeletal muscle inflammation and muscle physical characteristics and application thereof.

Background

Skeletal muscle is one of three major types of muscle in the human body, consisting of multinucleated contractile muscle cells (myofibers), accounting for about 40% of normal adult body weight. As a highly dynamic organ, skeletal muscle plays an important role in the motion, respiration, glycolipid metabolism and the like of a human body, and is an important supporting part for maintaining normal physiological functions of the human body and ensuring the quality of life. Inflammation plays a 'dual role' in skeletal muscle growth and development and repair after injury, and in the regeneration process after skeletal muscle injury, inflammation activates macrophages to inhibit inflammation, and simultaneously promotes muscle stem cell differentiation, angiogenesis and matrix remodeling, and promotes muscle regeneration and development, thus being an important means for enhancing muscle quality; however, when skeletal muscle is chronically stimulated by chronic inflammation, various pathological changes in skeletal muscle can occur, including muscle atrophy, muscle loss and insulin resistance of skeletal muscle (the main cause of type two diabetes). The occurrence and development of chronic inflammation of skeletal muscle is regulated by many factors, mainly including various chronic diseases, obesity and age-related aging, which seriously affect the quality of life of patients suffering from chronic diseases, obese and aging people. It is estimated that in the forty years to come, there will be more than two hundred million people that will be affected by muscle diseases [9]. Therefore, how to effectively control the inflammation of skeletal muscle, eliminate the adverse effect of chronic inflammation on skeletal muscle, and have important effects on maintaining body health and guaranteeing life quality.

At present, no specific medicine is used for treating the chronic inflammation of skeletal muscle at home and abroad, and the treatment means aiming at muscle inflammation mainly concentrate on non-steroidal anti-inflammatory drugs, antibiotics and partial Chinese patent medicines. For chronic inflammation-induced muscle diseases, the intervention of drugs such as testosterone, hormone, angiotensin converting enzyme inhibitor, creatine kinase and the like is mainly relied on to relieve muscle loss, but the drugs have limited functions and certain toxic and side effects. It has been found that catechin and theaflavine compounds, which are functional components in tea, are effective in promoting differentiation of skeletal muscle myoblasts, and in altering the muscle mechanical properties of mature skeletal muscle to maintain muscle health. In addition, catechin, theaflavins have been reported to regulate inflammatory response and inhibit the expression of inflammatory factors such as NF-kB, TNF- α, IL-1, IL-6, etc. In-vivo studies also find that polyphenol compounds in tea have good regulating effect on intestinal tract or liver inflammation induced by DSS, lipopolysaccharide and the like. Therefore, a compound which resists the chronic inflammation of skeletal muscle and has low side effect is searched from the functional components of the tea, and the compound has very important social significance and economic value in protecting skeletal muscle health and regulating related diseases caused by the chronic inflammation of skeletal muscle.

Currently, japanese prior art cn02157443.X discloses a muscle building agent which can be used for treating or preventing muscle degeneration, inflammation (especially arthritis) by its muscle building and anti-inflammatory actions; the main active ingredients of the technology are isoflavone and/or isoflavone glycoside and pungent substance, bitter substance and sour substance and cholic acid, and/or scymnol ester. The prior art CN201910753139.6 discloses a traditional Chinese medicine composition for treating bone joint and muscle pain; the formula and the weight portions of the components are as follows: 25-35 parts of astragalus membranaceus, 15-25 parts of rhizoma drynariae, 10-20 parts of peach kernel, 10-20 parts of safflower, 10-20 parts of rhizoma curcumae, 10-20 parts of radix salviae miltiorrhizae, 10-20 parts of radix dipsaci, 10-20 parts of cassia twig, 10-20 parts of radix angelicae pubescentis, 10-20 parts of radix sileris, 10-20 parts of dried orange peel, 1020 parts of notopterygium root, 10-20 parts of angelica sinensis, 10-20 parts of ligusticum wallichii, 10-20 parts of rhizoma corydalis, 15-25 parts of rhizoma atractylodis, 25-35 parts of rhizoma dioscoreae nipponicae and 10-20 parts of radix achyranthis bidentatae. The prior art CN200680037586.2 discloses a nutritional composition comprising a dairy product, in particular, the technology relates to a composition comprising whey growth factor extract, which extract is isolated from a dairy product by cation exchange chromatography, which may be used for reducing exercise-induced muscle inflammation.

Based on the above background and technical invention research reports, a great deal of research has demonstrated that natural ingredients in tea and Chinese herbal medicines have positive biological activities of regulating skeletal muscle inflammation. However, no specific application of theaflavins as a functional component in black tea in regulating skeletal muscle inflammation has been studied.

Disclosure of Invention

Aiming at the functional component theaflavine in the black tea, the inventor expands long-term research on the functional component theaflavine in the black tea, thereby completing the theaflavine nutrition formula with the functions of regulating skeletal muscle inflammation and muscle physical characteristics.

The invention aims to solve the technical problem of researching a theaflavine nutritional supplement capable of adjusting skeletal muscle inflammation and physical muscle characteristics. The invention discloses a formula of a nutritional supplement taking black tea as an auxiliary material, wherein the extract contains theaflavine with purity of 60% as a main raw material and catechin and polyester catechin mixture with purity of 40% as a residual raw material, and other natural traditional Chinese medicine extracts are added, and the specific regulation effect of the formula on skeletal muscle inflammation is clearly verified. The main problem of the invention is to solve the problems of preparing theaflavin with specific proportion and evaluating the actual effect of the combined formula.

In order to solve the technical problems, the invention obtains 60 percent of theaflavin extract from black tea soup by a separation and purification technology of a specific process, assists in preparing eucommia bark extract and polygonum cuspidatum extract as ingredients, and obtains a formula with a specific proportion. The main function of the formulation is to regulate skeletal muscle inflammation and muscle physical properties. And differentiated into mature myotubes in vitro using a C2C12 myoblast line, an in vitro myotube inflammation model was constructed by Lipopolysaccharide (LPS) induction, and the regulatory efficacy of the formulation on myotube inflammation was explored.

Preferably, the ratio of the 60% theaflavin mixture, the eucommia bark extract and the polygonum cuspidatum extract in the formula is 2:1:1, namely the 60% theaflavin mixture is taken as the main raw material and is twice as high as other auxiliary materials. Each formulation concentration was 60% theaflavin mixture (20. Mu.g/ml), eucommia ulmoides extract (10. Mu.g/ml), polygonum cuspidatum extract (10. Mu.g/ml) during the specific real-time course of the cytological experiments. The preparation of the medicine is scaled up when being used as a supplement conforming to human medicine.

The theaflavine nutrition mixture capable of regulating skeletal muscle inflammation and muscle physical characteristics is characterized in that: the theaflavine mixture with purity of 60% obtained by specific separation and purification is used as main raw materials, eucommia ulmoides and polygonum cuspidatum extracts are proportioned, and the main components for regulating skeletal muscle inflammation and muscle physical characteristics are theaflavine mixture with purity of 60%, eucommia ulmoides extracts and polygonum cuspidatum extracts according to the proportion of 2:1:1.

The mixture further comprises one or more of starch, cellulose, maltodextrin and oligosaccharide, more specifically 0.2-0.8 part of steviosin C, 20.2-0.8 part of vitamin D, 0.2-0.8 part of theanine and 5-10 parts of mineral salt.

In some embodiments, the mixture may be prepared in a dosage form selected from the group consisting of tablets, capsules, granules, or pills.

Furthermore, the invention also provides the application of the mixture in preparing a nutritional supplement for enhancing the movement characteristics of skeletal muscles.

Theaflavins are natural compounds with polyhydroxy and benzoazepinone structures formed between catechins under the enzymatic action of polyphenol oxidase during black tea processing, which are not only key compounds determining the quality components of black tea, but also called "soft gold" in tea. The theaflavin is polymerized from different catechin monomers and can be mainly divided into four monomers with different structures, namely theaflavin (THEAFLAVIN, TF), theaflavin-3-gallate (THEAFLAVIN-3-gallate, TF-3-G), theaflavin-3'-gallate (THEAFLAVIN-3' -gallate, TF-3 '-G) and theaflavin digallate (THEAFLAVIN-3, 3' -digallate, TFDG). Theaflavin is considered as a natural product with various beneficial biological functions, and has been widely reported to have remarkable effects in protecting human cardiovascular, resisting body inflammation and oxidative stress, resisting malignant tumor, regulating glycolipid metabolism, etc. Moreover, clinical studies have shown that theaflavine-rich black tea extract can promote recovery, reduce oxidative stress and delayed muscle soreness to acute anaerobic intervals, and help increase athlete movement frequency. In addition, clinically oral theaflavins have been found to reduce the proportion of body fat and increase the proportion of skeletal muscle, and it is interesting that theaflavins have better effects than catechins. In the present invention, the prepared theaflavine mixture was found to have a significant regulatory effect on LPS-induced skeletal muscle inflammation, which was found to significantly inhibit the expression of mRNA and protein levels of IL-6, IL-1, TNF-alpha in inflammatory myotubes.

The nutritional supplement provided by the invention has the beneficial effects that: the formula can effectively inhibit the expression of IL-6, IL-1 and TNF-alpha of inflammatory factors in skeletal myositis reaction, and has positive protective effect on the change of muscle mechanical characteristics under the inflammatory action. The formula is a formula consisting of natural product extracts and has the characteristics of natural products.

Drawings

Fig. 1: HPLC (high Performance liquid chromatography) detection of theaflavin purity;

fig. 2: LPS (40 mg/ml) induces elevated myotube ROS expression levels;

fig. 3: transcriptomics and bioinformatics analysis of the 60% theaflavin mixture formulation;

Fig. 4: modulation of mRNA levels of inflammatory factors TNF- α, NF-kB and IL-1 by a 60% theaflavin mixture formulation;

fig. 5: the 60% theaflavin mixture formula has the regulation effect on the protein level of inflammatory factors TNF-alpha, NF-kB and IL-1;

Fig. 6: the 60% theaflavin mixture formulation has regulating effect on mechanical properties of inflammatory myotubes.

Detailed Description

The present invention will be described in detail with reference to the following examples, but the scope of the present invention is not limited thereto.

EXAMPLE 60% theaflavin (60% theaflavin purity by HPLC) preparation of the mixture

200ML macroporous resin (HP-20, SP-70) of each model is accurately weighed, soaked in 95% ethanol for 24 hours, fully swelled, washed with distilled water until the washing liquid is non-milky, packed in a column by a wet method (column diameter-to-height ratio=5:15), and washed with water until the washing liquid is neutral for later use.

The specific use method is that different washing methods are used for macroporous resins of different types; separating tea Huang Sushi by using HP-20 macroporous resin, performing gradient elution by using 75% ethanol, collecting the solution with the action of 1h after elution, and freeze-drying to be detected; after SP-70 and 50% ethanol are eluted for half an hour, 75% ethanol is exchanged for continuous elution, the solution at 45mins is collected, and freeze-dried to be tested.

The final optimized process is that black tea: after water = 2:1, with a full soak of hot water for 15mins, the tea residue was filtered, loaded with macroporous resin of HP-20 at a flow rate of 1BV/h (bed volume per hour) and the effluent fraction was collected. And standing for 45min after the sample loading is finished. Eluting with 3BV (effective column volume) distilled water at a flow rate of 1BV/h, removing impurities with 2BV25% ethanol solution, eluting with 3BV 85% ethanol, collecting theaflavin-containing fraction, concentrating under reduced pressure in a rotary evaporator to recover ethanol completely, vacuum drying the concentrated extract, and quantitatively analyzing by HPLC to determine theaflavin content in the dried product to obtain theaflavin crude product.

Extracting and purifying theaflavin: dissolving the crude theaflavin product separated by column with 10 times of pure water, and extracting with ethyl acetate twice, each time with 0.6 times of pure water. Collecting extract fraction, and vacuum drying at 60deg.C to obtain theaflavin mixture.

The process can obtain theaflavin mixture with purity of 60% and most catechin mixture with residual 40% by HPLC (detection chart is shown in figure 1).

Example two evaluation of Effect of regulating skeletal muscle glucose absorption and mitochondrial production

Resveratrol has been reported to have excellent biological functions for skeletal muscle development and mitochondrial generation in a large number, and resveratrol used in experiments was purchased from Hunan Sanfu biotechnology limited, and the content detected by high performance liquid chromatography was 98%. Eucommia ulmoides is a traditional good herb for tonifying liver and kidney and strengthening tendons and bones. The specific preparation method of the eucommia ulmoides extract in the experiment is as follows: reflux-extracting with 10 times of pure water for 1 hr, collecting extractive solution, separating with polyamide column, eluting with 3BV50% ethanol, recovering ethanol from the eluate, and vacuum drying to obtain cortex Eucommiae extract with chlorogenic acid content of 50% detected by high performance liquid chromatography. A theaflavine nutritional formula for enhancing skeletal muscle movement characteristics was prepared as follows, and the raw materials were 2 parts of the extract prepared in example one and 1 part of resveratrol

And 1 part of eucommia ulmoides extract.

Example three evaluation of Effect of regulating skeletal muscle glucose absorption and mitochondrial production

After differentiation into mature myotubes by in vitro induction using C2C12 myoblasts as a model, the mature myotubes were incubated with LPS (40 mg/ml) for 24h to construct a myotube inflammation model.

Then, the inflammatory myotubes were treated with the formulation of example two, and the specific effect of the formulation of the present invention on myotube inflammation was clearly determined by detecting myotube inflammation level and mechanical properties using Q-PCR, ELISA, atomic force electron microscopy.

The specific experimental process is as follows:

1. preparation of various cell culture Medium

Cell culture medium: ① Proliferation medium (GM): 10% of fetal bovine serum+dmem high sugar medium; ② Differentiation Medium (DM): 2% horse serum + DMEM high sugar medium; ③ Contains 60% theaflavin (20 μg/ml) +eucommia bark extract (10 μg/ml) +polygonum cuspidatum extract (10 μg/ml): DM medium; ④ Differentiation medium (LPS) containing LPS: lps+dm medium.

2. Method for culturing in vitro myotubes and drug treatment

C2C12 cells were first cultured to 90% in 60mm dishes using GM medium, and then plated evenly at 3 x 106/well (six well plate) per well, 5x 105/well (12 well plate). After GM continued to be cultured to 80% -90%, DM medium was used to induce differentiation, fresh medium was changed daily until induced differentiation to fourth day, and mature myotubes were formed universally. The mature myotubes were then treated with lipopolysaccharide (LPS, 40 mg/ml) for 24h, an in vitro model of skeletal muscle chronic inflammation was constructed by measuring typical inflammatory factor expression levels, including IL-1, IL-6, TNF- α, and the effect of the formulation against muscle inflammation was determined by measuring the inflammatory factor expression levels, IL-1, IL-6, TNF- α, using a medium containing the formulation (i.e., DMEM high sugar medium, and adding 20. Mu.g/ml of a 60% theaflavine mixture, 10. Mu.g/ml of eucommia ulmoides extract, 10. Mu.g/ml of polygonum cuspidatum extract).

3. RNA extraction

(1) Target cells were collected, washed with 1×pbs, and lysed by adding an appropriate amount of Trizol according to the number of cells, and then placed on an ice box on a shaker;

(2) Blowing the cells after Trizol lysis in a culture dish by using a 1ml gun head, transferring the cells to a 1.5ml sterilized EP tube precooled on ice, standing for 3-5 minutes, adding precooled chloroform (Trizol: chloroform=5:1), fully mixing up and down, standing for 5 minutes on ice, then putting the cells in a high-speed refrigerated centrifuge, carrying out 12000 revolutions per minute, and centrifuging for 20 minutes;

(3) After centrifugation, absorbing the supernatant in the centrifuge tube to avoid sucking protein layer liquid, adding isopropyl alcohol after equal volume precooling, and placing on ice for 30 minutes or at-20 ℃ for permanent storage;

(4) Taking out the mixture of isopropanol and RNA, putting the mixture into a high-speed refrigerated centrifuge, centrifuging for half an hour at 7200 rpm, removing the supernatant, adding 75% precooled alcohol, flushing RNA precipitate, centrifuging for five minutes at 7200 rpm, and repeating the steps for more than two times;

(5) The EP tube is inverted, naturally dried for 7-10 minutes, cleaned with alcohol on the tube wall, and then added with a proper amount of enzyme-free water to detect the concentration of RNA.

4. Reverse transcription

According to the Norpran kit, preparing a reverse transcription system, namely, reversely transcribing target RNA into cDNA, adding 4 mu l of 4 XgDNA, adding 1000mg of target RNA, adding enzyme-free water to supplement the target RNA to a total volume of 16 mu l, putting the target RNA into a PCR instrument, and reacting for 2 minutes at 42 ℃; the sample was taken out, 4. Mu.l of 5X Surper Mix 2 was added, the PCR program was adjusted to 50℃for 15 minutes, 85℃for 5 seconds, and diluted 5-fold after the machine was started to prepare a working concentration.

5. Real-time fluorescent quantitative PCR

According to the procedure of the real-time fluorescence quantitative PCR kit of the Norflu, the following system is configured:

6. Detection of expression level of key genes such as inflammatory factors

QRT-PCR (quantitative real-time PCR) technology is an effective method for detecting gene transcription level at present; collecting all myotubes of an experimental group and a control group in an in-vitro experiment and total RNA of peripheral blood and tissue samples in an in-vivo experiment, and then carrying out reverse transcription to obtain cDNA for the next step of Q-PCR; cDNA obtained by reverse transcription was diluted five times, qRT-PCR was performed using MiniOpticon system (Bio-Rad, hercules, calif., USA) of SYBR Green (Applied Biological Materials), target molecules such as IL-1, IL-6, TNF- α, etc., were amplified, and finally qRT-PCR results were analyzed using a comparative Cycle Threshold (CT) method.

B. Enzyme-linked immunosorbent assay (ELISA) technology is an important means for detecting secreted proteins in peripheral blood; collecting myotube culture mediums under different treatment conditions, and detecting the content of inflammatory factor protein by using a qualitative and quantitative detection method for immune response by utilizing antigen-antibody specific binding. The specific method comprises the steps of collecting a cell culture medium, incubating with a specific antibody, performing a color reaction after overnight at 4 ℃, and detecting absorbance by using an enzyme-labeled instrument.

C. immunoblotting (Western Blotting) is a common detection method for researching the overall expression level of a protein, and the principle is that the specific combination of an antigen and an antibody is utilized, the primary antibody of a target protein is combined with the protein, the secondary antibody corresponding to the primary antibody is used for combination, and then the detection is carried out in a chemiluminescent instrument.

(1) Preparing SDS-polyacrylamide gel, adding 20-40mg of protein into each hole, adding protein mark at proper position, concentrating protein by using 80v voltage for 30min, separating protein by 120v separation voltage, and selecting beta-tublin as reference protein;

(2) Transferring: cutting off the upper layer glue; placing the lower gel into a membrane transferring liquid, and manufacturing a membrane transferring layer by using a sponge, filter paper and a membrane transferring clamp and a PVDF membrane; stabilizing the pressure for 100V, starting film transferring within 100min, and performing the whole film transferring process in ice;

(5) Closing: taking out PVDF membrane, and sealing at 4deg.C for 1 hr with prepared 5% skimmed milk;

(6) Incubating primary antibodies: separating target bands according to the size of target proteins and protein mark, adding target antibody working solution into a moisture preservation box, covering a cut film on the film, and incubating at 4 ℃. Taking out the membrane after overnight, and washing with PBST for 3 times each for 15min;

(7) Incubating a secondary antibody: secondary antibodies were formulated according to the instructions and incubated for 1h at 37 ℃ according to the primary antibody incubation method. PBST is washed for 3 times, each time for 15min;

(8) And (3) emitting light: and dripping ECL luminous liquid, and using WB imaging equipment to emit light. Recording according to Marker mark target protein;

(9) ImageJ data processing.

7. Myotube mechanical property detection

An atomic force microscope (atomic force microscope, AFM) AFM researches the surface structure and properties of a substance by detecting extremely weak atomic/molecular interaction force between the surface of a sample to be measured and a miniature force-sensitive element, and can image the surface of a measured object with high resolution.

The experiment uses AFM to characterize the morphology and mechanical properties of the cell surface, and detects the regulation effect of the formula of the invention on the mechanical properties of the inflammatory myotube surface. Briefly, after 2 washes with PBS, cells were fixed with 2% glutaraldehyde for 45 seconds, then with 4% polymethine solution for 20 minutes, finally washed with PBS more than 5 times, and finally with an appropriate amount of PBS for subsequent AFM scans. AFM imaging was performed using a JPK NanoWizard BioScience AFM (JPK Instr μ Ments, berlin, germany) mounted on a Nikon ECLIPSE TI2 (Nikon Corporation, tokyo, japan) inverted microscope, with at least 5 fixed cell images for five fields of view of each experimental sample by contact mode, all AFM scans were performed in fresh PBS buffer (pH 7.4) at room temperature.

AFM parameter setting: a V-probe of model HYDRA V-100NG (AppNano, CA, USA) 100 μm long was used to scan in liquid with a nominal spring constant of 0.292N/m. The force applied to the cantilever was manually adjusted to 50pN and the feedback gain was manually adjusted to obtain optimal resolution in height and deflection channels. Images were collected at a line scan rate of 0.3 to 0.6 Hz. AFM images were analyzed using JPK image processing software and surface mechanical data of 25 fixed cells scanned per sample were counted.

Experimental results

Earlier, LPS (40 mg/ml) was used to induce myotube inflammation in vitro, and after 24h of LPS treatment, the expression levels of inflammatory factors IL-1β, IL-6, tnf- α in myotubes were significantly elevated, and ROS levels in inflammatory myotubes were significantly elevated, demonstrating that LPS can induce myotubes in vitro to be in an inflammatory state (fig. 2& 3). Later, the present invention analyzed differential genes of inflammatory myotubes under the present formulation treatment by transcriptomic sequencing, found that of 229 genes significantly down-regulated in the 60% theaflavin formulation treatment group, which were significantly enriched in typical inflammatory pathways such as Jak-STAT, toll-like receptor signaling pathway, TNF signaling pathway, NF- κb signaling pathway, etc., which demonstrated that 60% of theaflavin formulation had an effect of inhibiting inflammatory pathways (fig. 3C).

To further demonstrate whether the formulations of the present invention have a difference in specific efficacy in inflammation prevention, inflammation treatment and inflammation reduction; by constructing three different models, ① TF1 and LPS are incubated together for 24 hours to simulate the resistance to inflammation; ② After LPS treatment of myotubes for 24 hours, TF1 is added for incubation for 24 hours, and the relief effect of TF1 on inflammation is simulated; ③ After TF1 pretreatment of myotubes for 24 hours, LPS was added to induce myotube inflammation, simulating the preventive effect of TF1 on skeletal muscle inflammation. The results show that TF1 showed resistance to myotube inflammation in both treatment modes ① and ②, including significant inhibition of IL-6, tnf- α in inflammatory myotubes at both mRNA and protein levels, but did not show significant inhibition of IL-1β expression (fig. 4& 5). In treatment regimen ③, TF1 did not show significant preventive effect on inflammatory factors. Based on this result, the 60% theaflavine mixture formulation of the present invention can significantly resist and alleviate skeletal muscle inflammation and also has a certain effect on preventing skeletal muscle inflammation (fig. 5).

In order to verify the regulation effect of the formula of the invention on the mechanical properties of the inflammatory myotubes, the mechanical properties of the inflammatory myotubes and normal myotubes are detected by atomic force electron microscopy, and the change of the rigidity and roughness of the myotubes surface caused by inflammation is found; and the cell rigidity and the roughness of the inflammatory myotubes treated by the 60% theaflavine formula are all reversed. This demonstrates that the 60% theaflavin formulation of the invention has some modulating effect on the surface mechanical properties of inflammatory skeletal muscle (figure 6).

Claims (5)

1. A theaflavin mixture for enhancing skeletal muscle movement characteristics, which is not used in foods or health products, characterized by comprising a theaflavin-containing black tea extract, eucommia bark extract, polygonum cuspidatum extract; the ratio of the black tea extract containing theaflavin to the eucommia bark extract to the polygonum cuspidatum extract is 2:1:1;

the extraction method of the black tea extract containing theaflavin comprises the following steps:

1) Fully soaking black tea in hot water for 10-20min, filtering tea residue, loading sample with macroporous resin of HP-20, and collecting effluent fraction; after the sample loading is finished, standing for 45min;

2) Eluting with 3BV distilled water with effective column volume, and collecting the eluent as water washing fraction;

3) Removing impurities by using a 25% ethanol solution with the effective column volume of 2BV, and collecting ethanol eluent;

4) Eluting with 3BV 85% ethanol with effective column volume, and collecting fraction containing theaflavin eluate;

5) Concentrating the eluent fraction obtained in the step 4) under reduced pressure until the solid content is more than 80%, and performing vacuum drying to obtain a crude product containing theaflavin;

6) Then extracting, adding ethyl acetate into the theaflavin crude product, and extracting for one to three times; collecting extract fraction, and vacuum drying to obtain final product containing theaflavin mixture 60%;

Wherein, in step 1), black tea: the hot water dosage ratio is 2:1, wherein the hot water temperature is 80-90 ℃; in 1), loading at a flow rate of 1 BV/h per hour of bed volume; and the macroporous resin of the HP-20 is soaked in 95% ethanol for 24h in advance, and is washed by distilled water after being fully swelled until the washing liquid is free from milky white, the column is packed by a wet method, and the diameter-to-height ratio of the column is 5:15, washing with water to be neutral for later use;

step 2), rinsing distilled water at a flow rate of 1 BV/h.

2. The theaflavin mixture according to claim 1, which is not used in foods or health products, wherein in step 6), crude theaflavin separated by column is dissolved with 10 times of pure water and extracted twice with ethyl acetate, each time with 0.5 to 0.7 times of pure water; extracting twice, collecting extract fraction, and vacuum drying at 60deg.C.

3. The theaflavin mixture according to any one of claims 1-2, which is not used in food or health products, wherein the mixture further comprises an auxiliary material selected from one or more of starch, cellulose, maltodextrin, oligosaccharides.

4. A theaflavin mixture as claimed in any one of claims 1 to 2 which is not used in food or health products wherein the mixture comprises 20 μg/ml of theaflavin-containing black tea extract, 10 μg/ml of eucommia bark extract and 10 μg/ml of polygonum cuspidatum extract.

5. The theaflavin nutrition mixture according to claim 4, which is not used in food or health products, wherein the mixture is formulated into a tablet, capsule, granule, or pill.