CN114468293A

CN114468293A - Theaflavin nutritional preparation for enhancing skeletal muscle movement characteristics and application thereof

Info

Publication number: CN114468293A
Application number: CN202210039369.8A
Authority: CN
Inventors: 刘子龙; 滕建造; 刘德时
Original assignee: Hunan Aijia Biotechnology Co ltd
Current assignee: Hunan Aijia Biotechnology Co ltd
Priority date: 2022-01-13
Filing date: 2022-01-13
Publication date: 2022-05-13

Abstract

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

Description

Theaflavin nutritional preparation for enhancing skeletal muscle movement characteristics and application thereof

Technical Field

The invention belongs to the field of health care products, and particularly relates to a theaflavin nutritional preparation capable of regulating skeletal muscle inflammation and muscle physical properties and application thereof.

Background

Skeletal muscle is one of the three major muscles of the human body and is composed of multinucleated contractile muscle cells (muscle fibers) that account for approximately 40% of normal adult body weight. As a highly dynamic organ, skeletal muscle plays an important role in human body movement, respiration, glycolipid metabolism and the like, and is an important supporting part for maintaining normal physiological functions of the human body and ensuring life quality. The inflammation plays a double role in the growth and development of skeletal muscle and the repair after injury, in the regeneration process after the skeletal muscle injury, the inflammation activates macrophages to inhibit the inflammation, simultaneously promotes the differentiation of muscle stem cells, angiogenesis and matrix remodeling, promotes the muscle regeneration and development, and is an important means for enhancing the muscle quality; however, when skeletal muscle is chronically stimulated by inflammation, various pathological changes in skeletal muscle can result, including muscle atrophy, muscle loss, and insulin resistance of skeletal muscle (the major cause of type ii diabetes). The occurrence and development of chronic inflammation of skeletal muscle are regulated by a plurality of factors, mainly including various chronic diseases, obesity and age-related aging, which seriously affect the quality of life of patients with chronic diseases, obese and aged people. It is estimated that over two hundred million people will be affected by muscle disease in the next forty years [9 ]. Therefore, how to effectively control the inflammation of skeletal muscle and eliminate the adverse effect of chronic inflammation on skeletal muscle plays an important role in maintaining the health of the organism and guaranteeing the quality of life.

At present, no specific medicine is available at home and abroad for treating the chronic inflammation of skeletal muscle, and the treatment means aiming at the muscle inflammation mainly focuses on non-steroidal anti-inflammatory drugs, antibiotics and partial Chinese patent medicines. For muscle diseases induced by chronic inflammation, the muscle loss is relieved mainly by the intervention of drugs such as testosterone, hormone, angiotensin converting enzyme inhibitor, creatine kinase and the like, but the drugs have limited functions and certain toxic and side effects. The research shows that the catechin and the theaflavin compound which are functional components in the tea can effectively promote the differentiation capability of skeletal myoblasts, change the mechanical characteristics of muscles of mature skeletal muscles and keep the muscle healthy. Besides, catechin and theaflavin compounds are reported to regulate inflammatory reaction and inhibit the expression of inflammatory factors such as NF-kB, TNF-alpha, IL-1, IL-6 and the like. In-vivo research also finds that the polyphenol compounds in the tea have good regulating effect on intestinal tract or liver inflammation induced by DSS, lipopolysaccharide and the like. Therefore, a compound with low side effect for resisting skeletal muscle chronic inflammation is searched from the functional components of the tea, and the compound has very important social significance and economic value in protecting the skeletal muscle health and regulating related diseases caused by the skeletal muscle chronic inflammation.

At present, the Japanese prior art CN02157443.X discloses a strong muscle agent that can be used for treating or preventing muscle degeneration, inflammation (especially arthritis) action by its muscle strengthening 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 parts of the components are as follows: 25-35 parts of astragalus membranaceus, 15-25 parts of drynaria baronii, 10-20 parts of peach kernel, 10-20 parts of safflower, 10-20 parts of curcuma zedoary, 10-20 parts of salvia miltiorrhiza, 10-20 parts of teasel root, 10-20 parts of cassia twig, 10-20 parts of radix angelicae pubescentis, 10-20 parts of divaricate saposhnikovia root, 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. Prior art CN200680037586.2 discloses a dietary nutritional composition comprising a dairy product, in particular the art relates to the use of a composition comprising whey growth factor extract, which is isolated from a dairy product by cation exchange chromatography, for reducing exercise-induced muscle inflammation.

Based on the above background and the reports of the research of the technical invention, a great deal of research has proved that the natural ingredients in tea leaves and Chinese herbal medicines have positive biological activity of regulating skeletal muscle inflammation. However, no research has been carried out on the specific application of the functional component theaflavin in black tea in regulating the inflammation of skeletal muscle.

Disclosure of Invention

Aiming at the functional component theaflavin in the black tea, the invention has wide application prospect, and the inventor develops long-term research on the functional component theaflavin, thereby completing the theaflavin nutritional formula with the functions of regulating the skeletal muscle inflammation and the muscle physical property.

The invention aims to solve the technical problem of researching a theaflavin nutritional supplement capable of regulating skeletal muscle inflammation and muscle physical properties. The invention discloses a nutritional supplement formula which takes extract extracted from black tea (wherein the extract contains theaflavin with the purity of 60% as a main dietary raw material, and the rest 40% is catechin and polyester catechin mixture) and is added with other natural traditional Chinese medicine extracts as auxiliary materials for the first time, 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 preparation of the theaflavin with a specific proportion and evaluation of the actual effect of the combined formula.

In order to solve the technical problems, 60% of theaflavin extract is obtained from black tea soup by a separation and purification technology of a specific process, and a formula with a specific proportion is obtained by taking an eucommia ulmoides extract and a polygonum cuspidatum extract as auxiliary materials. The formula has the main function of regulating skeletal muscle inflammation and muscle physical properties. And using the differentiation of the C2C12 myoblast cell line into mature myotubes in vitro, constructing an in vitro myotube inflammation model through Lipopolysaccharide (LPS) induction, and exploring the regulation effect of the formula on myotube inflammation.

Preferably, the ratio of the 60% theaflavin mixture, the eucommia ulmoides extract and the polygonum cuspidatum extract in the formula is 2:1:1, namely, the theaflavin mixture with the purity of 60% is used as a main raw material and is twice of that of other auxiliary materials. The concentrations of the formulations in the real-time course of cytology were 60% theaflavin mixture (20. mu.g/ml), eucommia ulmoides extract (10. mu.g/ml) and Polygonum cuspidatum extract (10. mu.g/ml). The dietary supplement is prepared according with human pharmacokinetics and is enlarged in proportion.

The invention provides a theaflavin nutrient mixture capable of regulating skeletal muscle inflammation and muscle physical characteristics, which is characterized in that: the method is characterized in that a theaflavin mixture with the purity of 60% obtained by specific separation and purification is used as a main raw material, eucommia ulmoides and polygonum cuspidatum extract are matched according to the ratio of 2:1:1, and the main components for regulating skeletal muscle inflammation and muscle physical characteristics are the theaflavin mixture with the purity of 60%, the eucommia ulmoides extract and the polygonum cuspidatum extract.

The mixture further contains adjuvants selected from one or more of starch, cellulose, maltodextrin, and oligosaccharide, more specifically steviosin C0.2-0.8 parts, vitamin D20.2-0.8 parts, theanine 0.2-0.8 parts, and mineral salt 5-10 parts.

In some embodiments, the mixture can be prepared into a dosage form selected from a tablet, capsule, granule, or pill.

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

Theaflavin is a natural compound with polyhydroxy and benzophenone structures formed between catechins under the enzymatic action of polyphenol oxidase in the processing process of black tea, is not only a key compound determining the quality components of black tea, but also is called 'soft gold' in tea leaves. Theaflavin is polymerized by 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 is widely reported to have remarkable efficacies in the aspects of protecting human cardiovascular system, resisting inflammation and oxidative stress of organisms, resisting malignant tumors, regulating glycolipid metabolism and the like. Moreover, clinical studies have shown that black tea extract rich in theaflavins can promote recovery, reduce oxidative stress and delayed muscle soreness during acute anaerobic periods, and contribute to increase in athlete's exercise frequency. In addition, it has been found that clinical oral administration of theaflavins decreases the proportion of body fat and increases the proportion of skeletal muscle, and interestingly theaflavins are more potent than catechins. In the invention, the prepared theaflavin mixture is found to have a remarkable regulation effect on the skeletal muscle inflammation induced by LPS, and the expression of mRNA and protein levels of IL-6, IL-1 and TNF-alpha in inflammatory myotubes can be remarkably inhibited.

The dietary nutrition 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 protection effect on the change of muscle mechanical properties under the action of inflammation. The formula is a dietary formula consisting of natural product extracts, and has natural product properties.

Drawings

FIG. 1: detecting the purity of theaflavin by HPLC;

FIG. 2 is a schematic diagram: LPS (40 mg/ml) induced an increase in myotube ROS expression levels;

FIG. 3: transcriptomics and bioinformatics analysis of 60% theaflavin mixture formula;

FIG. 4: the regulation effect of a 60% theaflavin mixture formula on the mRNA level of inflammatory factors TNF-alpha, NF-kB and IL-1;

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

FIG. 6: the regulation effect of the 60% theaflavin mixture formula on the 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-preparation of a 60% theaflavin (60% theaflavin purity by HPLC)

Accurately weighing 200 mL of macroporous resin (HP-20, SP-70) of each type, soaking in 95% ethanol for 24h, fully swelling, washing with distilled water until the washing liquid is opalescent, packing with a wet method (column diameter-height ratio =5: 15), and washing with water to neutrality for later use.

The specific using method is that different washing methods are used for different types of macroporous resin; when the HP-20 macroporous resin is used for separating theaflavin, 75% ethanol is used for gradient elution, a solution which acts for 1 hour after elution is collected, and the solution is freeze-dried to be tested; and SP-70, after eluting with 50% ethanol for half an hour, changing 75% ethanol for continuous elution, collecting the solution at the position of 45mins, and freeze-drying to be tested.

The final optimized process is as follows: water =2:1, after soaking in hot water for 15 mins, the tea residue was filtered, loaded with HP-20 macroporous resin at a flow rate of 1 BV/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) of distilled water at a flow rate of 1 BV/h, removing impurities with 2 BV of 25% ethanol solution, eluting with 3BV of 85% ethanol, collecting theaflavin-containing fraction, concentrating under reduced pressure in a rotary evaporator to completely recover ethanol, vacuum drying the concentrated extract, and quantitatively analyzing and determining the theaflavin content in the dried product by HPLC to obtain a crude theaflavin product.

And (3) extracting and purifying theaflavin: dissolving the theaflavin crude product separated by the column with 10 times of pure water, and adding ethyl acetate for extraction twice, wherein the dosage of each time is 0.6 times of the volume of the pure water. Collecting extract fraction, and vacuum drying at 60 deg.C to obtain theaflavin mixture with specific ratio.

The process can obtain a theaflavin mixture with a purity of 60% as determined by HPLC, and the remaining 40% is mostly catechin mixture (the detection chart is shown in figure 1).

EXAMPLE two evaluation of the Effect of regulating glucose uptake into skeletal muscle and mitochondrial production

Resveratrol is reported to have good biological functions on skeletal muscle development and mitochondrial generation, experimental resveratrol is purchased from three-fortune biotechnology limited in Hunan, and the content of resveratrol detected by high performance liquid chromatography is 98%. Eucommia bark is a traditional good medicine 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: extracting with 10 times of pure water under reflux for 1 hr, collecting extractive solution, separating with polyamide column, eluting with 3BV50% ethanol, recovering ethanol from eluate, vacuum drying to obtain Eucommiae cortex extract, and detecting chlorogenic acid content by high performance liquid chromatography to 50%. The theaflavin nutritional formula for enhancing the skeletal muscle motor characteristics is prepared according to the following formula, and the raw materials comprise 2 parts of the extract prepared in the first embodiment, 1 part of resveratrol and 1 part of eucommia ulmoides extract.

EXAMPLE III evaluation of the Effect of regulating glucose uptake into skeletal muscle and mitochondrial Generation

A myotube inflammation model was constructed by inducing differentiation of C2C12 myoblasts into mature myotubes in vitro and incubating the mature myotubes with LPS (40 mg/ml) for 24 h.

Then the inflammatory myotubes are treated by using the formula of the second example, the level and the mechanical property of the myotube inflammation are detected by using Q-PCR, ELISA and an atomic force electron microscope, and the specific action of the formula of the invention on the myotube inflammation is clearly judged.

The specific experimental process is as follows:

1. preparation of various cell culture media

Cell culture medium: [ proliferation Medium (GM): 10% fetal bovine serum + DMEM high-glucose medium; ② Differentiation Medium (DM): 2% horse serum + DMEM high-sugar medium; ③ 60 percent of theaflavin (20 mu g/ml), eucommia bark extract (10 mu g/ml), giant knotweed rhizome extract (10 mu g/ml): DM culture medium; (iv) LPS-containing differentiation medium (LPS): LPS + DM medium.

Culture method and drug treatment of external myotubes

C2C12 cells were first cultured to 90% in a 60mm dish using GM medium, and then inoculated uniformly into cell culture plates at 3X 106/well (six well plates), 5X 105/well (12 well plates) per well. And (3) continuously culturing the GM to 80-90%, using a DM culture medium to induce differentiation, and replacing a fresh culture medium every day until the mature myotubes are generally formed by the fourth day of induced differentiation. Then, mature myotubes were treated with lipopolysaccharide (LPS, 40 mg/ml) for 24h, a model of chronic inflammation of skeletal muscle in vitro was constructed by measuring the expression levels of typical inflammatory factors including IL-1, IL-6, TNF- α, and the effects of the formulation against muscle inflammation were determined by measuring the expression levels of the inflammatory factors, IL-1, IL-6, TNF- α, and treating the inflammatory myotubes with a medium containing the formulation (i.e., DMEM high-glucose medium with the addition of 20. mu.g/ml of a 60% theaflavin mixture, 10. mu.g/ml of eucommia ulmoides extract, 10. mu.g/ml of Polygonum cuspidatum extract).

Extraction of

(1) Collecting target cells, washing the cells with 1 XPBS, adding a proper amount of Trizol according to the number of the cells, and then placing the cells on an ice box on a shaker for cracking;

(2) blowing and beating the cells after Trizol lysis in a culture dish by using a 1ml gun head, transferring the cells into a 1.5ml sterilized EP tube precooled on ice, standing for 3-5 minutes, adding precooled trichloromethane (Trizol: trichloromethane =5: 1), fully and uniformly mixing the cells up and down, standing for 5 minutes on ice, then putting the mixture into a high-speed refrigerated centrifuge, rotating at 12000 rpm, and centrifuging for 20 minutes;

(3) after centrifugation, sucking the supernatant in a centrifuge tube to avoid sucking protein layer liquid, adding isopropanol after equal volume precooling, and placing on ice for 30 minutes or permanently preserving at-20 ℃;

(4) taking out the mixture of the isopropanol and the RNA, centrifuging for half an hour after 7200 r/min in a high-speed freezing centrifuge, removing the supernatant, adding 75% precooled alcohol, washing the RNA precipitate, centrifuging for five minutes at 7200 r/min, and repeating the steps for more than two times;

(5) inverting the EP tube, naturally airing for 7-10 minutes, cleaning alcohol on the tube wall, adding a proper amount of enzyme-free water, and detecting the RNA concentration.

Reverse transcription

According to the Novozan kit, a reverse transcription system is configured, target RNA is reversely transcribed into cDNA, 4 mu l of 4 Xg DNA is added, 1000mg of target RNA is added, enzyme-free water is added to supplement the total volume to 16 mu l, and the mixture is placed into a PCR instrument and reacts for 2 minutes at 42 ℃; the sample was removed, 4. mu.l of 5 XSurrer Mix 2 was added, the PCR program was adjusted to 50 ℃ for 15 minutes and 85 ℃ for 5 seconds, and the sample was diluted 5-fold after unloading to prepare the working concentration.

Real-time fluorescent quantitative PCR

According to the Novozan real-time fluorescent quantitative PCR kit program, the following system is configured:

reagent	Dosage of
		SYBR Green	5μl
Forward and reverse primers	1μl
		Template cDNA
	2 μl
		Nuclease-free water	To 10. mu.l

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

a. The qRT-PCR (quantitative real-time PCR) technology is an effective method for detecting the gene transcription level at present; collecting total RNA of all myotubes of an experimental group and a control group in an in vitro experiment and peripheral blood and tissue samples in an in vivo experiment, and then carrying out reverse transcription to obtain cDNA for the next Q-PCR; the cDNA obtained by reverse transcription was diluted five times, qRT-PCR was performed using the 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 the results of qRT-PCR were analyzed using the comparative Cycle Threshold (CT) method.

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

c. The immunoblotting (Western Blotting) technique is a common detection method for studying the overall expression level of protein, and the principle is to utilize the specific binding of antigen and antibody, use the primary antibody of the target protein to bind with protein, use the secondary antibody corresponding to the primary antibody to bind, and then detect in a chemiluminescence instrument.

(1) Preparing SDS-polyacrylamide gel, adding 20-40mg of protein into each hole, adding protein mark at a proper position, firstly using 80 v voltage to concentrate protein for 30 minutes, then using 120 v separation voltage to separate protein on separation gel, and selecting beta-tublin as internal reference protein;

(2) film transfer: cutting off the upper layer glue; putting the lower layer gel into a membrane transferring liquid, and preparing a membrane transferring layer by using a sponge, filter paper and a membrane transferring clamp and a PVDF membrane; stabilizing the pressure at 100V, starting to rotate the film within 100 min, and performing the whole film rotating process in ice;

(5) and (3) sealing: taking out the PVDF membrane, and sealing with prepared 5% skimmed milk at 4 deg.C for 1 h;

(6) incubating the primary antibody: separating target bands according to the size of target protein and protein mark, adding target antibody working solution into a moisture preservation box, covering the cut membrane on the target antibody working solution, and incubating at 4 ℃. After overnight, the membranes were removed and washed 3 times with PBST, 15 min each time;

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

(8) luminescence: ECL luminous liquid is dripped, and WB imaging equipment is used for emitting light. Marking target protein according to a Marker and making a record;

(9) and ImageJ data processing.

Myotube mechanical property detection

An Atomic Force Microscope (AFM) studies the surface structure and properties of a substance by detecting a very weak atomic/molecular interaction force between the surface of a sample to be measured and a micro force sensitive element, and can image the surface of the measured object with high resolution.

AFM is used for representing the surface appearance and mechanical properties of cells in the experiment, and the regulation effect of the formula on the mechanical properties of the surface of the inflammatory myotube is detected. Briefly, after 2 washes with PBS, cells were fixed with 2% glutaraldehyde for 45 seconds, then with 4% methanolic solution for 20 minutes, finally washed with PBS for more than 5 times, and finally with appropriate amounts of PBS for subsequent AFM scans. AFM imaging was performed using JPK NanoWizard 4 BioScience AFM (JPK Instr μ Ments, Berlin, Germany) mounted on a Nikon Eclipse Ti2 (Nikon Corporation, Tokyo, Japan) inverted microscope, imaging at least 5 fixed cells in five fields of view for each experimental sample by contact mode, all AFM scans being performed in fresh PBS buffer (pH 7.4) at room temperature.

AFM parameter setting: scanning was performed in liquid using a 100 μm long V-shaped probe of type HYDRA6V-100NG (AppNano, CA, USA) 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 the best resolution in height and deflection channel. Images were collected at a line scan rate of 0.3 to 0.6 Hz. AFM images were analyzed using JPK image processing software to count the surface mechanical data for 25 fixed cells scanned for each sample.

Results of the experiment

In the early stage, in vitro myotube inflammation was induced using LPS (40 mg/ml), and 24h after LPS treatment of myotubes, the expression levels of inflammatory factors IL-1 β, IL-6, TNF- α in myotubes were significantly increased and the ROS level in inflammatory myotubes was significantly increased, demonstrating that LPS can induce the inflammatory state of the extra myotubes (fig. 2& 3). Then, the invention analyzes the differential genes of the inflammatory myotubes under the treatment of the formula through transcriptomic sequencing, and finds that the 60% theaflavin formula treatment group is obviously enriched in Jak-STAT, Toll-like receptor signal pathway, TNF signal pathway, NF-kB signal pathway and other typical inflammatory pathways among 229 genes which are obviously reduced, thereby indicating that 60% theaflavin formula has the function of inhibiting the inflammatory pathways (figure 3C).

To further demonstrate whether the formulations of the present invention differ in specific efficacy in inflammation prevention, inflammation treatment and inflammation relief; through constructing three different models, TF1 and LPS are incubated for 24h, and the resistance effect on inflammation is simulated; adding TF1 for incubation for 24h after the muscular tube is treated by LPS for 24h, and simulating the relieving effect of TF1 on inflammation; and thirdly, after the TF1 pretreats the myotubes for 24 hours, LPS is added to induce myotube inflammation, and the prevention effect of TF1 on skeletal muscle inflammation is simulated. The results show that TF1 showed resistance to myotube inflammation in both treatment regimes (r) and (r), including significant inhibition of IL-6, TNF- α in inflammatory myotubes at both mRNA and protein levels, but not of IL-1 β expression (fig. 4& 5). In treatment mode c, TF1 did not exhibit significant prophylactic effects on inflammatory factors. Based on the results, the 60% theaflavin mixture formulation of the present invention can significantly resist and relieve the inflammation of skeletal muscle, and has certain effect on preventing the inflammation of skeletal muscle (fig. 5).

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

Claims

1. A method for extracting a black tea extract containing theaflavin for enhancing skeletal muscle movement characteristics is characterized by comprising the following steps:

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

2) eluting with 3BV distilled water of effective column volume, and collecting eluate as water eluate fraction;

3) removing impurities with 2 BV25% ethanol solution of effective column volume, and collecting ethanol eluate;

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

5) vacuum drying the eluent fraction obtained in the step 4) when the solid content is over 80 percent after vacuum concentration to obtain a crude product containing theaflavin;

6) then extracting, adding ethyl acetate into the crude theaflavin product, and extracting for one to three times; collecting the extract fraction and vacuum drying.

2. A process for the extraction of a black tea extract with enhanced skeletal muscle motor properties as claimed in claim 1 wherein the final product is controlled to contain 60% theaflavin mixture.

3. A process for the extraction of black tea extract for enhancing skeletal muscle motor characteristics as claimed in claim 1), wherein in 1) the ratio of black tea: the using ratio of hot water is 2:1, wherein the hot water is 80-90 ℃; preferably, in 1), the sample is loaded at a flow rate of 1 BV/h per hour of bed volume; and the HP-20 macroporous resin is soaked in 95% ethanol for 24h in advance, fully swelled and washed by distilled water until the washing liquid is milk white, and then wet-packed into a column (preferably, the height ratio of the column diameter =5: 15) and washed by water until the washing liquid is neutral for later use.

4. A process for extracting black tea extract for enhancing skeletal muscle motor characteristics as claimed in claim 1, wherein in step 2), distilled water is eluted at a flow rate of 1 BV/h per hour for 3 bed volumes of column.

5. The method for extracting black tea extract with skeletal muscle motor enhancing effect as claimed in claim 1, wherein in step 6), the crude theaflavins separated by column chromatography are dissolved in 10 times of pure water, and ethyl acetate is added in an amount of 0.5-0.7 times of the volume of the mixture; extracting twice, collecting extract fraction, and vacuum drying at 60 deg.C.

6. A theaflavin nutritional blend for enhancing skeletal muscle motor characteristics comprising an extract of black tea containing theaflavins, an extract of eucommia ulmoides, and an extract of polygonum cuspidatum prepared by the method of any one of claims 1 to 6.

7. The mixture according to claim 7, wherein the ratio of the theaflavin-containing black tea extract, the eucommia ulmoides extract, and the polygonum cuspidatum extract is 2:1: 1; preferably, the black tea extract containing theaflavin is 20 μ g/ml, the eucommia bark extract is 10 μ g/ml and the giant knotweed extract is 10 μ g/ml.

8. The mixture according to claim 6 or 7, further comprising one or more adjuvants selected from starch, cellulose, maltodextrin, and oligosaccharide, more specifically 0.2-0.8 parts of steviosin C, 20.2-0.8 parts of vitamin D, 0.2-0.8 parts of theanine, and 5-10 parts of mineral salt.

9. The mixture of claim 8, wherein the formulation is in a dosage form selected from the group consisting of a tablet, a capsule, a granule, and a pill.

10. Use of a mixture according to any one of claims 6 to 9 in the manufacture of a dietary nutritional supplement for enhancing skeletal muscle motor characteristics.