Traditional Chinese medicine composition for resisting stress and improving meat quality for pigs, and preparation method and application thereof

Abstract

The application discloses a traditional Chinese medicine composition for resisting stress and improving meat quality for pigs and a preparation method and application thereof. The traditional Chinese medicine composition is prepared from the following raw materials in parts by mass: Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chou 15-45 parts, Mimosa pudica L. 10-30 parts, Galeopsis segetum Benth. 10-50 parts, Glycyrrhiza uralensis Fisch. 10-20 parts, Acanthopanax 5-40 parts and Kadsura longipedunculata 10-45 parts. The preparation method comprises the following steps: 1) preparing Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chou water extract, Mimosa pudica L. ethanol extract, Galeopsis segetum Benth. ethanol solution extract, Glycyrrhiza uralensis Fisch. water extract, Acanthopanax ethanol solution extract and Kadsura longipedunculata water extract respectively; and 2) uniformly mixing the extracts to prepare the traditional Chinese medicine composition. The traditional Chinese medicine composition is reasonably composed, and according to the effective component characteristics of different traditional Chinese medicines, different extraction methods are used to improve the extraction rate of the effective components and guarantee the production and application effect of the traditional Chinese medicine composition.

Description

Technical Field

[0001] This invention belongs to the field of feed additives, specifically relating to a traditional Chinese medicine composition for pigs to resist stress and improve meat quality, its preparation method and application. Background Technology

[0002] With the upgrading of consumption, the public's demand for pork quality continues to rise, and improving quality and efficiency has become the mainstream direction for the future development of the pig industry. Meat quality is affected by multiple factors, including slaughtering, transportation, diet, and feed additives. In the later stages of pig fattening, pigs are prone to various stress reactions such as transportation, high temperature, and slaughter, which not only cause a decrease in feed intake and a slowdown in daily weight gain, but also significantly deteriorate pork quality, leading to obvious differences in product quality.

[0003] Currently, there is a lack of specialized conditioning products and application solutions for stress reduction and meat quality improvement in pigs. Traditional Chinese medicine (TCM) possesses multiple effects, including antiviral, sedative, and calming properties, demonstrating significant advantages in regulating physiological functions and enhancing overall immunity. Furthermore, TCM is derived from natural sources, is green and safe, and does not leave drug residues in pork or pigs after feeding, meeting the requirements for healthy livestock farming and food safety. Its application is highly safe and reliable. Summary of the Invention

[0004] The purpose of this invention is to provide a traditional Chinese medicine composition for swine stress relief and / or meat quality improvement, and its preparation method. This traditional Chinese medicine composition can be fully utilized by pigs to achieve the purpose of stress relief and meat quality improvement, and has no toxic side effects.

[0005] The traditional Chinese medicine composition for anti-stress and / or meat quality improvement in pigs provided by the present invention is made from the following raw materials in parts by weight: 15-45 parts of Ziziphus jujuba, 10-30 parts of Mimosa pudica, 10-50 parts of Clematis armandii, 10-20 parts of Glycyrrhiza uralensis, 5-40 parts of Acanthopanax senticosus and 10-45 parts of Celastrus orbiculatus fruit.

[0006] In some embodiments of the present invention, the traditional Chinese medicine composition is made from the following raw materials in parts by weight: 20-35 parts of Ziziphus jujuba, 20-30 parts of Mimosa pudica, 25-35 parts of Clematis armandii, 10-15 parts of Glycyrrhiza uralensis, 30-40 parts of Acanthopanax senticosus, and 25-45 parts of Celastrus orbiculatus fruit.

[0007] In one specific embodiment of the present invention, the traditional Chinese medicine composition is made from the following raw materials in parts by weight: 35 parts of Chinese jujube, 20 parts of mimosa, 25 parts of fragrant grass, 15 parts of licorice, 30 parts of Acanthopanax senticosus, and 45 parts of Celastrus orbiculatus fruit.

[0008] In some embodiments of the present invention, the traditional Chinese medicine composition includes water extract of Ziziphus jujuba, ethanol extract of Mimosa pudica, ethanol solution extract of Clematis armandii, water extract of Glycyrrhiza uralensis, ethanol solution extract of Acanthopanax senticosus, and water extract of Celastrus orbiculatus fruit.

[0009] The method for preparing the traditional Chinese medicine composition for swine stress prevention and / or meat quality improvement provided by the present invention includes the following steps: 1) Prepare aqueous extracts of Ziziphus jujuba, ethanol extracts of Mimosa pudica, ethanol extracts of Clematis armandii, aqueous extracts of Glycyrrhiza uralensis, ethanol extracts of Acanthopanax senticosus, and aqueous extracts of Celastrus orbiculatus fruit, respectively; 2) Mix the above extracts evenly to obtain the traditional Chinese medicine composition.

[0010] In some embodiments of the present invention, the preparation method of the Chinese jujube water extract is as follows: the dried Chinese jujube medicinal material is pulverized and passed through a 20-40 mesh sieve, 6-14 times the amount of water is added, and it is extracted 1-3 times, 1-3 hours each time, at an extraction temperature of 60-100℃. The extract is filtered through a membrane, the extracts are combined, concentrated under reduced pressure at 60-80℃ to a relative density of 1.0-1.2, and spray-dried to obtain the Chinese jujube extract.

[0011] In some embodiments of the present invention, the preparation method of the mimosa ethanol extract is as follows: the dried mimosa herb is pulverized and passed through a 20-40 mesh sieve, 9-14 times the amount of anhydrous ethanol is added, and the extract is extracted 2-3 times, 1-4 hours each time, at an extraction temperature of 65-100℃. The extract is filtered through a membrane, the extracts are combined, concentrated under reduced pressure at 50-80℃ to a relative density of 1.0-1.2, and spray-dried to obtain the mimosa extract.

[0012] In some embodiments of the present invention, the preparation method of the ethanol extract of *Hedyotis diffusa* is as follows: the dried *Hedyotis diffusa* herb is pulverized and passed through a 20-40 mesh sieve, 6-12 times the volume of 60%-80% ethanol solution is added, and the mixture is refluxed for extraction 2-3 times, 1-3 hours each time, at a reflux temperature of 60-80°C. The reflux liquid is filtered through a membrane, the reflux liquids are combined, and the mixture is concentrated under reduced pressure at 60-80°C to a relative density of 1.10-1.20. The mixture is then spray-dried to obtain the *Hedyotis diffusa* extract.

[0013] In some embodiments of the present invention, the preparation method of the licorice water extract is as follows: the dried licorice herb is pulverized and passed through a 20-40 mesh sieve, 8-14 times the amount of water is added, and the extract is extracted 1-4 times, 1-3 hours each time, at an extraction temperature of 85-100℃. The extract is filtered through a membrane, the extracts are combined, and the extract is concentrated under reduced pressure at 60-80℃ to a relative density of 1.0-1.2. The extract is then spray-dried to obtain the licorice extract.

[0014] In some embodiments of the present invention, the preparation method of the Acanthopanax senticosus ethanol solution extract is as follows: the dried Acanthopanax senticosus herb is pulverized and passed through a 20-40 mesh sieve, 6-12 times the volume fraction of 60%-80% ethanol solution is added, and the mixture is refluxed for extraction 2-3 times, 1-3 hours each time, at a reflux temperature of 60-80°C. The reflux liquid is filtered through a membrane, the reflux liquids are combined, and the mixture is concentrated under reduced pressure at 60-80°C to a relative density of 1.10-1.20. The mixture is then spray-dried to obtain the Acanthopanax senticosus extract.

[0015] In some embodiments of the present invention, the preparation method of the aqueous extract of Celastrus orbiculatus fruit is as follows: the dried Celastrus orbiculatus fruit is pulverized and passed through a 20-40 mesh sieve, 6-14 times the amount of water is added, and the mixture is extracted 1-3 times, 1-3 hours each time, at an extraction temperature of 60-100℃. The extract is filtered through a membrane, the extracts are combined, and the mixture is concentrated under reduced pressure at 60-80℃ to a relative density of 1.0-1.2. The mixture is then spray-dried to obtain the Celastrus orbiculatus fruit extract.

[0016] The present invention also provides the application of the above-mentioned traditional Chinese medicine composition in the preparation of feed or feed additives for pigs that are designed to resist stress and / or improve meat quality.

[0017] In some embodiments of the present invention, the amount of the traditional Chinese medicine composition added is 0.1-0.5% of the total mass of the feed.

[0018] In one specific embodiment of the present invention, the amount of the traditional Chinese medicine composition added is 0.2% of the total mass of the feed.

[0019] In some embodiments of the present invention, the pig is a fattening pig.

[0020] The present invention also provides the application of the above-mentioned traditional Chinese medicine composition in the preparation of anti-stress and / or meat quality-improving preparations for pigs.

[0021] In some embodiments of the present invention, the method of using the preparation is as follows: adding the traditional Chinese medicine composition to the feed for fattening pigs; the amount of the preparation is 0.1-0.5% of the total mass of the feed.

[0022] In some embodiments of the present invention, the pig is a fattening pig.

[0023] Compared with the prior art, the present invention has the following beneficial technical effects: 1. The traditional Chinese medicine composition of the present invention has a reasonable formulation, and according to the characteristics of the effective components of different traditional Chinese medicines, different extraction methods are used to improve the extraction rate of the effective components and ensure the production and application effect of the traditional Chinese medicine composition.

[0024] 2. The traditional Chinese medicine combination of the present invention, when added to the diet of fattening pigs, has the effect of resisting stress and improving meat quality.

[0025] 3. This product is a pure combination of traditional Chinese medicines, which is safe, has no toxic side effects, and does not produce drug resistance. Attached Figure Description

[0026] Figure 1 Images showing the measurement of dripping water loss.

[0027] Figure 2 The flesh color rating scale is as follows: 1. Pale grayish-pink to white, 1 point; 2. Grayish-pink, 2 points; 3. Bright red or bright red, 3 points; 4. Dark red, 4 points; 5. Purplish-red, 5 points; 6. Dark purplish-red, 6 points.

[0028] Figure 3 The image shows a flesh color rating chart, where 0 is the control; 1 is sample 1; 2 is sample 2; 3 is sample 3; 4 is sample 4; 5 is sample 5; 6 is sample 6; 7 is sample 7; 8 is sample 8; 9 is sample 9; and 10 is sample 10.

[0029] Figure 4-15 The liquid chromatography chromatogram for the determination of inosinic acid content in pork is shown below. Figure 4 This is the chromatogram of inosinic acid reference standard. Figure 5 For blank groups, Figure 6 For sample 1, Figure 7 For sample 2, Figure 8 For sample 3, Figure 9 Sample 4, Figure 10 Sample 5, Figure 11 Sample 6, Figure 12 Sample 7, Figure 13 Sample 8, Figure 14 Sample 9, Figure 15 Sample 10.

[0030] Figure 16-18 Images showing the induction of myogenesis in C2C12 cells, in which... Figure 16 These are normal C2C12 cells. Figure 17 To induce 2-day C2C12 cells, Figure 18 To induce 3-day C2C12 cells. Detailed Implementation

[0031] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0033] Example 1 A traditional Chinese medicine composition for stress relief and meat quality improvement in fattening pigs is prepared from the following raw materials in parts by weight: 15 parts of Ziziphus jujuba, 20 parts of Mimosa pudica, 15 parts of Citrus aurantium, 10 parts of Glycyrrhiza uralensis, 5 parts of Acanthopanax senticosus, and 25 parts of Celastrus orbiculatus fruit.

[0034] The preparation method of the traditional Chinese medicine composition is as follows: Southern jujube extract: The dried southern jujube medicinal material was pulverized and passed through a 40-mesh sieve. 10 times the amount of water was added, and the extract was extracted twice, 2 hours each time, at an extraction temperature of 100℃. The extract was filtered through a membrane, and the extracts were combined and concentrated under reduced pressure at 65℃ to a relative density of 1.2. The extract was then spray-dried to obtain southern jujube extract. Mimosa extract: The dried mimosa herb was pulverized and passed through a 20-mesh sieve. 10 times the amount of anhydrous ethanol was added, and the mixture was extracted twice, 2 hours each time, at an extraction temperature of 80℃. The extract was filtered through a membrane, and the extracts were combined and concentrated under reduced pressure at 60℃ to a relative density of 1.2. The extract was then spray-dried to obtain mimosa extract. Fragrant grass extract: The dried fragrant grass herb was pulverized and passed through a 40-mesh sieve. 12 times the volume fraction of 80% ethanol solution was added, and the mixture was refluxed and extracted 3 times, 1.5 h each time, at a reflux temperature of 60℃. The reflux liquid was filtered through a membrane, and the reflux liquids were combined and concentrated under reduced pressure at 65℃ to a relative density of 1.18. The mixture was then spray-dried to obtain fragrant grass extract. Licorice extract: The dried licorice herb was pulverized and passed through a 40-mesh sieve. 12 times the amount of water was added, and the extract was extracted twice, 3 hours each time, at an extraction temperature of 100℃. The extract was filtered through a membrane, and the extracts were combined and concentrated under reduced pressure at 80℃ to a relative density of 1.2. The extract was then spray-dried to obtain licorice extract. Acanthopanax senticosus extract: The dried Acanthopanax senticosus herb was pulverized and passed through a 40-mesh sieve. 10 times the volume fraction of 60% ethanol solution was added, and the mixture was refluxed and extracted 3 times, 1.5 hours each time, at a reflux temperature of 60℃. The reflux liquid was filtered through a membrane, and the reflux liquids were combined and concentrated under reduced pressure at 65℃ to a relative density of 1.15. The mixture was then spray-dried to obtain Acanthopanax senticosus extract. Celastrus orbiculatus fruit extract: The dried Celastrus orbiculatus fruit was pulverized and passed through a 40-mesh sieve. 11 times the amount of water was added, and the extract was extracted twice, 1.5 hours each time, at an extraction temperature of 100℃. The extract was filtered through a membrane, and the extracts were combined and concentrated under reduced pressure at 60℃ to a relative density of 1.2. The extract was then spray-dried to obtain Celastrus orbiculatus fruit extract. A traditional Chinese medicine composition was prepared by uniformly mixing extracts of Chinese jujube, mimosa, fragrant grass, licorice, eleutherococcus senticosus, and clematis fruit with excipients.

[0035] Example 2 A traditional Chinese medicine composition for anti-stress and meat quality improvement in fattening pigs is prepared from the following raw materials in parts by weight: 20 parts of Chinese jujube, 25 parts of mimosa, 10 parts of fragrant grass, 10 parts of licorice, 10 parts of Acanthopanax senticosus, and 10 parts of Celastrus orbiculatus fruit.

[0036] The preparation method of the above-mentioned traditional Chinese medicine composition for anti-stress and improvement of meat quality in fattening pigs is the same as in Example 1.

[0037] Example 3 A traditional Chinese medicine composition for anti-stress and meat quality improvement in fattening pigs is prepared from the following raw materials in parts by weight: 35 parts of Chinese jujube, 20 parts of mimosa, 25 parts of fragrant grass, 15 parts of licorice, 30 parts of Acanthopanax senticosus, and 45 parts of Celastrus orbiculatus fruit.

[0038] The preparation method of the above-mentioned traditional Chinese medicine composition for anti-stress and improvement of meat quality in fattening pigs is the same as in Example 1.

[0039] Example 4 A traditional Chinese medicine composition for anti-stress and meat quality improvement in fattening pigs is prepared from the following raw materials in parts by weight: 15 parts of Ziziphus jujuba, 10 parts of Mimosa pudica, 15 parts of Citrus aurantium, 15 parts of Glycyrrhiza uralensis, 5 parts of Acanthopanax senticosus, and 30 parts of Celastrus orbiculatus fruit.

[0040] The preparation method of the above-mentioned traditional Chinese medicine composition for anti-stress and improvement of meat quality in fattening pigs is the same as in Example 1.

[0041] Example 5 A traditional Chinese medicine composition for anti-stress and meat quality improvement in fattening pigs is prepared from the following raw materials in parts by weight: 15 parts of Chinese jujube, 15 parts of mimosa, 15 parts of fragrant grass, 10 parts of licorice, 15 parts of Acanthopanax senticosus, and 20 parts of Celastrus orbiculatus fruit.

[0042] The preparation method of the above-mentioned traditional Chinese medicine composition for anti-stress and improvement of meat quality in fattening pigs is the same as in Example 1.

[0043] Example 6 A traditional Chinese medicine composition for stress relief and meat quality improvement in fattening pigs is prepared from the following raw materials in parts by weight: 20 parts of Chinese jujube, 10 parts of mimosa, 20 parts of fragrant grass, 20 parts of licorice, 20 parts of Acanthopanax senticosus, and 30 parts of Celastrus orbiculatus fruit.

[0044] The preparation method of the above-mentioned traditional Chinese medicine composition for anti-stress and improvement of meat quality in fattening pigs is the same as in Example 1.

[0045] Example 7 A traditional Chinese medicine composition for anti-stress and meat quality improvement in fattening pigs is prepared from the following raw materials in parts by weight: 25 parts of Ziziphus jujuba, 20 parts of Mimosa pudica, 10 parts of Citrus aurantium, 20 parts of Glycyrrhiza uralensis, 5 parts of Acanthopanax senticosus, and 30 parts of Celastrus orbiculatus fruit.

[0046] The preparation method of the above-mentioned traditional Chinese medicine composition for anti-stress and improvement of meat quality in fattening pigs is the same as in Example 1.

[0047] Example 8 A traditional Chinese medicine composition for anti-stress and meat quality improvement in fattening pigs is prepared from the following raw materials in parts by weight: 30 parts of Chinese jujube, 10 parts of mimosa, 35 parts of fragrant grass, 10 parts of licorice, 5 parts of Acanthopanax senticosus, and 10 parts of Celastrus orbiculatus fruit.

[0048] The preparation method of the above-mentioned traditional Chinese medicine composition for anti-stress and improvement of meat quality in fattening pigs is the same as in Example 1.

[0049] Implementation Method 9 Prepared according to the method requirements of patent number CN201410017211.6, specifically as follows: 10 parts refined yam powder, 4 parts dried plum powder, 5 parts licorice powder, 5 parts astragalus powder, 6 parts wolfberry powder, 5 parts Acanthopanax senticosus powder, 2 parts tea polyphenols, 8 parts microecological preparation, 3 parts compound vitamins, 3 parts compound enzyme preparation, 1 part lysine, 0.6 parts methionine, 0.4 parts arginine, and the remaining carrier, which is composed of zeolite powder, defatted rice bran, and wheat bran in a mass ratio of 2:4:1. Implementation method 9.

[0050] Example 10 A traditional Chinese medicine composition for anti-stress and improvement of meat quality in fattening pigs, characterized by different mass ratios of traditional Chinese medicine: 15 parts of Ziziphus jujuba, 10 parts of Mimosa pudica, 40 parts of Clematis armandii, 10 parts of Glycyrrhiza uralensis, 5 parts of Acanthopanax senticosus, and 20 parts of Celastrus orbiculatus fruit.

[0051] The preparation method of the above-mentioned traditional Chinese medicine composition for anti-stress and improvement of meat quality in fattening pigs is the same as in Example 1.

[0052] Effect verification: (1) Effect of the sample on antioxidant stress of C2C12 myocytes I. Preparation of Reagents and Materials 1. Preparation of basal culture medium Preparation of cell culture medium: 89% DMEM high glucose medium + 10% fetal bovine serum + 1% penicillin antibiotics, as shown in Table 1.

[0053] Table 1

[0054] 2. Other key reagents PBS buffer: used for washing cells.

[0055] Trypsin digestion solution: The literature uses conventional trypsin. Cryopreservation solution: The standard cell cryopreservation solution is usually: 70% complete culture medium + 20% FBS + 10% DMSO.

[0056] 3. Instrument Environment Incubator: 37℃, 5% CO2, saturated humidity.

[0057] Microscope: An inverted microscope used to observe cell morphology and density.

[0058] Centrifuge: Centrifuge at room temperature, speed set to 1000 rpm.

[0059] Water bath: 37℃ constant temperature, used for cell resuscitation.

[0060] 4. Sample Screening The selected samples are shown in Table 2.

[0061] Table 2

[0062] II. Experimental Procedure 1. Cell Thawing Applicable scenario: Retrieving frozen cells from a liquid nitrogen tank for initial culture.

[0063] Preheating: Prepare a 37°C constant temperature water bath and preheated complete culture medium in advance.

[0064] Removal of cryovials: Quickly remove the cryovials from the liquid nitrogen.

[0065] Thawing: Immediately place the cryovials in a 37°C water bath and shake them constantly to thaw them quickly.

[0066] Key point: Act quickly until the ice crystals in the cryovial completely disappear (about 1-2 minutes) to avoid prolonged low temperature damage to cells.

[0067] Transfer: In a clean bench, disinfect the outer wall of the cryovial by wiping it with alcohol. Transfer the cell suspension into a 15 ml centrifuge tube containing 2 ml of complete culture medium.

[0068] Centrifugation: After mixing, place in a centrifuge and centrifuge at 1000 rpm for 3 min.

[0069] Remove supernatant: Carefully discard the supernatant (to remove DMSO), being careful not to aspirate the cell precipitate at the bottom.

[0070] Resuspension: Add 4 ml of fresh complete culture medium, gently pipette to mix, and resuspend the cell pellet.

[0071] Inoculation: Transfer the cell suspension to a culture flask (T25 or T75, depending on the cell quantity), and add an appropriate amount of culture medium to the appropriate total volume (e.g., the total liquid volume of a T25 flask is usually 5-6 ml).

[0072] Culture: Incubate in a 37℃, 5% CO2 incubator.

[0073] Observation: The cell status (adhesion, morphology, and presence of contamination) was observed for the first time after 24 hours.

[0074] 2. Routine cell observation and medium change Observation frequency: Observe daily under an inverted microscope.

[0075] Observation indicators: Morphology: The cells are fibroblast-like or myoblast-like, spindle-shaped or polygonal, and have good refractive properties.

[0076] Density: Focus on cell confluence.

[0077] Status: The color of the culture medium (yellow indicates vigorous metabolism or contamination), and the presence or absence of particulate floating matter (signs of contamination).

[0078] Medium change: If the cells are not fully grown and the culture medium turns yellow, the complete culture medium can be changed every 2-3 days.

[0079] When changing the medium, remove the old medium, wash gently with PBS (optional), and add fresh, preheated medium.

[0080] 3. Cell passage (subculturing) Triggering condition: Passage is performed when the cell confluence reaches 80%-90%.

[0081] Preparation: Preheat PBS, trypsin, and complete culture medium to room temperature or 37°C. Sterilize under UV light in a laminar flow hood for 30 minutes.

[0082] Discard the culture medium: Remove the culture flask and aspirate the old complete culture medium.

[0083] Washing: Add an appropriate amount of PBS buffer, gently agitate to rinse the cell surface, and discard the PBS. Repeat this step 3 times (to remove residual serum and prevent serum from inhibiting trypsin activity).

[0084] Digestion: Add 2 ml of trypsin digestion solution (enough to cover the bottom of the flask). Incubate at 37°C for 3 minutes (the exact time needs to be monitored under a microscope; the standard is 3 minutes).

[0085] Microscopic monitoring: Remove the culture flask and observe it under a microscope.

[0086] End point determination: Digestion should be stopped immediately when the intercellular spaces widen, cells retract and become rounded, and some cells begin to detach. Note: Do not over-digest, as this will affect cell viability.

[0087] To terminate digestion: immediately add 2 ml of complete culture medium (containing serum, which can neutralize trypsin).

[0088] Dispersion by pipetting: Use a pipette or pipette to draw up the culture medium and gently pipette the bottom of the culture flask to completely detach the adherent cells and form a single-cell suspension. The pipetting motion should be gentle to avoid generating too many air bubbles that could damage the cells.

[0089] Collection and centrifugation: Transfer the cell suspension to a 15 ml centrifuge tube. Centrifuge at 1000 rpm for 3 min.

[0090] Resuspension: Discard the supernatant. Add 4 ml of fresh complete culture medium and gently pipette to resuspend the cell pellet, ensuring even dispersion.

[0091] Subculturing: Subculture the culture into two separate flasks according to experimental requirements (usually at a 1:2 or 1:3 ratio). For example, divide 4 ml of suspension into two new culture flasks and add culture medium to each flask to the appropriate volume.

[0092] Culture: Shake the culture flask well (using the "cross method") and place it in a 37℃, 5% CO2 incubator for further culture.

[0093] III. Test Methods Phase 1: Cell Seeding and Adhesion Digestion and Counting: Take cells from passage 5 in logarithmic growth phase, covering 80%-90% of a T25 culture flask. Remove the culture medium from the flask, add 3 ml of PBS, and gently pipette three times using a 1 ml pipette. Repeat this process twice. Add 2 ml of trypsin and incubate at 37°C for 1-2 minutes. When the cell edges become shiny and the cells become rounded, immediately add 2 ml of serum-containing culture medium to stop the digestion. Centrifuge at 1000 rpm for 5 minutes. Transfer the supernatant to a waste container and add 5 ml of fresh culture medium. Gently resuspend the cells by pipetting.

[0094] For cell counting, aspirate 0.9 ml of sterile PBS into a 10 ml centrifuge tube, add 0.1 ml of resuspended cells, gently mix with a 1 ml pipette, aspirate 10 μL onto a hemocytometer, gently cover with a coverslip, and observe the cell count under a 10x microscope. Dilute as needed.

[0095] Cell concentration (cells / mL) = (Total number of cells in four large squares / 4) × Dilution factor × 10 4 Inoculation density: 96-well plate (hemocytometer counting): density 1×10⁻⁶ 5 The seeding volume per well is typically 100 μL (approximately 1 × 10⁻⁶ cells / mL), with a cell / mL ratio. 4 (cells).

[0096] For adherent culture: Before placing the T25 culture flask, wipe the entire surface of the flask, your hands, and the area around the incubator door with 75% alcohol swabs to prevent contamination. Incubate at 37°C with 5% CO2 for 24 hours until the cells are fully adhered.

[0097] Phase 2: Induction of myoblasts Culture: Use a flat-bottomed 96-well plate to seed cells until the cells have grown to more than 80% of the 96-well plate.

[0098] Induction: Add 100 μL of 2% horse serum + 97% DMEM high glucose + 1% antibiotic medium to each well. Change the culture medium every 2 days. Induce for 3 days.

[0099] Judgment criteria: Cell fusion: Originally spindle-shaped individual myoblasts will approach each other, align, and fuse.

[0100] Myotube formation: The fused cells form long, tubular, multinucleated structures called myotubes.

[0101] Nucleus arrangement: Multiple nuclei within the myotube exhibit a unique "beaded" arrangement.

[0102] Phase 3: Grouping and Drug Administration (Core Modeling Steps) 3.1 Hydrogen peroxide modeling Hydrogen peroxide solution dilution: First, pipette 9.09 ml of PBS into a 15 ml centrifuge tube. Then, pipette 0.91 ml of 3% hydrogen peroxide solution into the same centrifuge tube and mix thoroughly. Next, pipette 9 ml of PBS into the 15 ml centrifuge tube, add 1 ml of the mixed solution, and mix thoroughly again. Then, add 9 ml of DMEM high-glucose medium to the 15 ml centrifuge tube, and add 1 ml of the second mixed solution. Mix thoroughly to obtain a 0.8 mmol / L hydrogen peroxide solution. Then, dilute to concentrations of 0.1, 0.2, and 0.4 mmol / L using the isostatic dilution method (prepare fresh before use).

[0103] Transfer 2% horse serum culture medium from a 96-well plate to a waste container. Add hydrogen peroxide solutions of 0, 0.1, 0.2, 0.4, and 0.8 mmol / L in sequence. Add DMEM high-glucose medium in a 0-gradient manner, increasing the concentration from low to high. Add 100 μL of hydrogen peroxide dilution to each well. After addition, wipe the surface of the 96-well plate with 75% ethanol. Incubate at 37°C for 30 min. Then, transfer the hydrogen peroxide medium to a waste container in the same order of addition. Wash twice with 100 μL of PBS each time, gently transfer to the waste container, add 100 μL of 2% horse serum medium, add 10 μL of CCK-8, and incubate at 37°C for 1 h. Measure the absorbance at 450 nm.

[0104] Phase IV Validation Trial Sample preparation: Accurately weigh 150 mg of extract from each formulation, dissolve it in 10 ml of distilled water or ethanol according to the extraction solvent, and prepare a sample concentration of 15 mg / ml according to the formulation ratio. Filter the solution through a 0.22 µm filter to sterilize and set aside for use. Dilute to a concentration of 150 µg / ml in 2% horse serum culture medium solution according to the 10-fold dilution method.

[0105] Following the method in Table 2, add 100 μL of drug-containing culture medium to each well. Add 2% horse serum culture medium solution to the blank group and the model group, and incubate in a cell culture incubator for 24 h.

[0106] Aspirate the culture medium from the 96-well plate into a waste container and wash twice with PBS. The blank control group was added to DMEM high-glucose medium; the other groups were cultured in 800 µmol / L hydrogen peroxide medium at 37°C for 30 min according to the modeling method described in 3.1. Then, aspirate the culture medium into the waste container in the order of addition. Wash twice with 100 μL of PBS each time, gently aspirate into the waste container, add 100 μL of 2% horse serum medium and 10 μL of CCK-8, and incubate at 37°C for 1 h. Measure the absorbance at 450 nm.

[0107] IV. Results 4.1 Induction of C2C12 cells into myotube cells Please see details Figure 16-18 Cell fusion: Individual myoblasts, originally spindle-shaped, approach, align, and fuse together. Myotube formation: The fused cells form elongated, tubular, multinucleated structures called myotubes. Nucleus arrangement: The multiple nuclei within the myotube exhibit a unique "beaded" arrangement.

[0108] 4.2 Hydrogen peroxide modeling test The results of the hydrogen peroxide modeling experiment are shown in Table 3. Hydrogen peroxide solutions of 100, 200, 400, and 800 µmol / L all significantly reduced cell viability, with the cell viability at a concentration of 800 µmol / L being significantly lower than that of the other groups.

[0109] Table 3. Hydrogen peroxide modeling test (n=6)

[0110] 4.3 Drug Screening Trial The results of antioxidant stress of samples 1-10 are shown in Table 4. The cell viability of each group was significantly higher than that of the model group, and the cell viability of samples 3, 7 and 8 was significantly higher than that of sample 9.

[0111] Table 4. Screening results of hydrogen peroxide stress models for samples 1-10

[0112] Five conclusions In summary, samples 3, 7, and 8 exhibit significant antioxidant stress effects, with sample 3 showing the best antioxidant stress effect.

[0113] (2) Effects of the sample on improving meat quality in fattening pigs 1. Experimental Objective Traditional Chinese medicine compositions that can improve meat quality were screened out.

[0114] 2. Experimental Materials The experimental materials are shown in Table 5.

[0115] Table 5. Amount of Traditional Chinese Medicine Added to Feed

[0116] 3. Test Methods 3.1 Experimental Grouping 495 piglets weighing 60kg were divided into 11 treatment groups, with three replicates per group and 15 piglets per replicate. The piglets were fed from 60kg to 120kg at slaughter. They had free access to feed and water throughout the process. The control group was fed a basal diet, while the experimental groups were fed a combination of traditional Chinese medicine and basal diet.

[0117] 3.2 Feeding and Management Before the experiment began, the pigsty was thoroughly cleaned and disinfected, including all equipment used during the experiment. Normal feeding and management resumed. 3.3 Meat quality index testing Drip loss test: Cut the sample into pieces with a length * width * height of 2 cm * 2 cm * 1 cm. Weigh the meat pieces on a balance and record the weight as W1. Then, with the muscle fibers pointing vertically downwards, thread a thin wire through one end of the meat strip and suspend it in a plastic bag (the meat sample should not touch the bag wall). Seal the bag with a rubber band and hang it in a refrigerator at 4 ℃ for 24 hours. Then, remove the meat strip, wipe off any residual liquid, and weigh it (W2). Each meat sample was tested in triplicate, and the average weight was recorded as the drip loss. The drip loss measurement diagram is shown below. Figure 1 As shown.

[0118]

[0119] In the formula: W1 is the mass (g) of the meat sample before hanging, and W2 is the mass (g) of the meat sample after hanging for 24 hours.

[0120] A drip loss of 1.5%-5% is considered normal meat; a drip loss >5% is considered PSE meat; and a drip loss <1.5% is considered DFD meat.

[0121] Flesh color detection: Place the sample in a backlit area for flesh color scoring. See Table 6 for scoring criteria details. Figure 2 .

[0122] Two samples were evaluated for each sample, and the color of flesh 1 and flesh 2 were recorded.

[0123] Two samples were evaluated for each sample, and the color of flesh 1 and flesh 2 were recorded.

[0124] Table 6. Flesh Color Scoring Criteria

[0125] Note: A flesh color score of 1 point indicates PSE flesh; The flesh color score is 2 points, which is close to PSE flesh. A flesh color score of 3-4 indicates normal flesh color. The flesh color score is 5 points, which is close to that of DFD meat; The flesh color score is 6 points, indicating it is DFD meat.

[0126] Flesh color rating = (Flesh color 1 + Flesh color 2) / 2 Inosinic acid content detection: Weigh 5.00g of chopped pork sample into a 50mL centrifuge tube, add 20mL of 1mol / L hydrochloric acid solution, and vortex to mix for 1min.

[0127] Extract using ultrasound for 20 minutes (ultrasound power set to 250W), during which the centrifuge tubes can be shaken appropriately.

[0128] Centrifuge the centrifuge tubes at 8000 r / min for 10 min and collect the supernatant.

[0129] Adjust the pH of the supernatant to 6.5-7.0 with a 1 mol / L sodium hydroxide solution (add dropwise while stirring, and measure accurately with a pH meter with an accuracy of ±0.01).

[0130] Centrifuge again at 8000 rpm for 10 min, collect the supernatant, filter it through a 0.45 μm microporous membrane, and set aside for later use. If necessary, the filtrate can be further purified by passing it through a C18 solid-phase extraction column. Activate the column with 5 mL of ultrapure water and 5 mL of 100% methanol, load the sample, rinse with 5 mL of ultrapure water, and finally elute with 3 mL of 100% methanol. Collect the eluent, dry it under nitrogen, and then dilute it to 1 mL with the mobile phase (composition shown in the chromatographic conditions section).

[0131] 5-Inosinic acid standard stock solution: Accurately weigh 10 mg of 5-inosinic acid standard into a 50 mL volumetric flask, dissolve it in ultrapure water and dilute to the mark to prepare a standard stock solution with a concentration of 0.2 mg / mL (i.e. 200 μg / mL).

[0132] Chromatographic conditions Chromatographic column: C18 reversed-phase column (4.6mm × 250mm, particle size 5μm).

[0133] Mobile phase: Phase A was a 0.05 mol / L potassium dihydrogen phosphate solution (pH adjusted to 4.0 with phosphoric acid), and Phase B was methanol (purity ≥99.9%). Gradient elution was used: 0-5 min, 95% (v / v) A + 5% (v / v) B; 5-15 min, 90% (v / v) A + 10% (v / v) B; 15-25 min, 80% (v / v) A + 20% (v / v) B; 25-30 min, 95% (v / v) A + 5% (v / v) B.

[0134] Flow rate: 1.0 mL / min.

[0135] Column temperature: 30℃.

[0136] Detection wavelength: 5-inosinic acid 254 nm. Injection volume: 20 μL.

[0137] Sample Measurement Inject the prepared pork sample solution into a high-performance liquid chromatograph (HPLC) and analyze it according to the chromatographic conditions set above. Record the peak area and calculate the content.

[0138] 4. Data Analysis The experimental data were analyzed using SPSS 20.0 statistical software. A p-value less than or equal to 0.05 indicated a significant difference between groups, while a p-value less than or equal to 0.10 indicated a trend in the difference between groups.

[0139] 5 Results 5.1 Meat quality test results The meat quality test results are shown in Table 7 and Figure 3 As shown, in terms of meat color score, the control group tended to be PSE meat, while the meat color scores of samples 2, 3, and 7 were significantly higher than those of the control group ( P <0.05); Regarding drip loss, samples 3 and 7 were significantly lower than the control group ( P <0.05).

[0140] Table 7 Meat quality test results

[0141] 5.2 Inosinic acid content detection The inosinic acid content of pork samples is shown in Table 8. Figure 4-16 As shown, sample group 3 was the highest, 27.21% higher than the control group; followed by samples 7 and 8, which were 20.55% and 16.85% higher than the control group, respectively. Sample 9 was 4.36% lower than the control group.

[0142] Table 8. Inosinic acid content (%)

[0143] 6. Conclusion In summary, sample 3 significantly improved meat color score, significantly reduced drip loss, increased inosinic acid content, and enhanced umami flavor. Therefore, sample 3 has anti-stress and meat quality-improving effects.

[0144] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A traditional Chinese medicine composition for swine to resist stress and / or improve meat quality, made from the following raw materials in parts by weight: 15-45 parts of Ziziphus jujuba, 10-30 parts of Mimosa pudica, 10-50 parts of Clematis armandii, 10-20 parts of Glycyrrhiza uralensis, 5-40 parts of Acanthopanax senticosus and 10-45 parts of Celastrus orbiculatus fruit.

2. The traditional Chinese medicine composition for swine stress relief and / or meat quality improvement according to claim 1, characterized in that: The traditional Chinese medicine composition is made from the following raw materials in parts by weight: 20-35 parts of Ziziphus jujuba, 20-30 parts of Mimosa pudica, 25-35 parts of Clematis armandii, 10-15 parts of Glycyrrhiza uralensis, 30-40 parts of Acanthopanax senticosus, and 25-45 parts of Celastrus orbiculatus fruit.

3. The traditional Chinese medicine composition for swine stress prevention and / or meat quality improvement according to claim 1 or 2, characterized in that: The traditional Chinese medicine composition is made from the following raw materials in parts by weight: 35 parts of Chinese jujube, 20 parts of mimosa, 25 parts of fragrant grass, 15 parts of licorice, 30 parts of Acanthopanax senticosus, and 45 parts of Celastrus orbiculatus fruit.

4. The traditional Chinese medicine composition for swine stress prevention and / or meat quality improvement according to any one of claims 1-3, characterized in that: The traditional Chinese medicine composition includes water extracts of Ziziphus jujuba, ethanol extracts of Mimosa pudica, ethanol extracts of Clematis armandii, water extracts of Glycyrrhiza uralensis, ethanol extracts of Acanthopanax senticosus, and water extracts of Celastrus orbiculatus fruit.

5. A method for preparing the traditional Chinese medicine composition according to any one of claims 1-4, comprising the following steps: 1) Prepare aqueous extracts of Ziziphus jujuba, ethanol extracts of Mimosa pudica, ethanol extracts of Clematis armandii, aqueous extracts of Glycyrrhiza uralensis, ethanol extracts of Acanthopanax senticosus, and aqueous extracts of Celastrus orbiculatus fruit, respectively; 2) Mix the above extracts evenly to obtain the traditional Chinese medicine composition.

6. The preparation method according to claim 5, characterized in that: The specific preparation method of the Chinese jujube water extract is as follows: the dried Chinese jujube medicinal material is pulverized and passed through a 20-40 mesh sieve, 6-14 times the amount of water is added, and it is extracted 1-3 times, 1-3 hours each time, at an extraction temperature of 60-100℃. The extract is filtered through a membrane, the extracts are combined, and concentrated under reduced pressure at 60-80℃ to a relative density of 1.0-1.

2. The extract is then spray-dried to obtain the Chinese jujube extract. And / or, the specific preparation method of the mimosa ethanol extract is as follows: the dried mimosa herb is pulverized and passed through a 20-40 mesh sieve, 9-14 times the amount of anhydrous ethanol is added, and it is extracted 2-3 times, 1-4 hours each time, at an extraction temperature of 65-100℃. The extract is filtered through a membrane, the extracts are combined, concentrated under reduced pressure at 50-80℃ to a relative density of 1.0-1.2, and spray-dried to obtain the mimosa extract; And / or, the specific preparation method of the *Xiangpai* ethanol extract is as follows: the dried *Xiangpai* herb is pulverized and passed through a 20-40 mesh sieve, 6-12 times the volume of 60%-80% ethanol solution is added, and the mixture is refluxed for extraction 2-3 times, 1-3 hours each time, at a reflux temperature of 60-80℃. The reflux liquid is filtered through a membrane, the reflux liquids are combined, and the mixture is concentrated under reduced pressure at 60-80℃ to a relative density of 1.10-1.

20. The mixture is then spray-dried to obtain the *Xiangpai* extract. And / or, the specific preparation method of the licorice water extract is as follows: the dried licorice herb is pulverized and passed through a 20-40 mesh sieve, 8-14 times the amount of water is added, and it is extracted 1-4 times, 1-3 hours each time, at an extraction temperature of 85-100℃. The extract is filtered through a membrane, the extracts are combined, concentrated under reduced pressure at 60-80℃ to a relative density of 1.0-1.2, and spray-dried to obtain the licorice extract; And / or, the specific preparation method of the Acanthopanax senticosus ethanol solution extract is as follows: the dried Acanthopanax senticosus herb is pulverized and passed through a 20-40 mesh sieve, 6-12 times the volume fraction of 60%-80% ethanol solution is added, and the mixture is refluxed for extraction 2-3 times, 1-3 hours each time, at a reflux temperature of 60-80℃. The reflux liquid is filtered through a membrane, the reflux liquids are combined, and the mixture is concentrated under reduced pressure at 60-80℃ to a relative density of 1.10-1.

20. The mixture is then spray-dried to obtain the Acanthopanax senticosus extract. And / or, the specific preparation method of the aqueous extract of Celastrus orbiculatus fruit is as follows: the dried Celastrus orbiculatus fruit is pulverized and passed through a 20-40 mesh sieve, 6-14 times the amount of water is added, and it is extracted 1-3 times, 1-3 hours each time, at an extraction temperature of 60-100℃. The extract is filtered through a membrane, the extracts are combined, and concentrated under reduced pressure at 60-80℃ to a relative density of 1.0-1.

2. The extract is then spray-dried to obtain the Celastrus orbiculatus fruit extract.

7. The use of the traditional Chinese medicine composition according to any one of claims 1-4 in the preparation of feed or feed additives for pigs that are designed to resist stress and / or improve meat quality.

8. The application according to claim 7, characterized in that: The amount of the traditional Chinese medicine composition added is 0.1-0.5% of the total feed mass; And / or, the pig is a fattening pig.

9. The use of the traditional Chinese medicine composition according to any one of claims 1-4 in the preparation of a preparation for swine to resist stress and / or improve meat quality.

10. The application according to claim 9, characterized in that: The method of using the preparation is as follows: add the traditional Chinese medicine composition to the feed for fattening pigs; the dosage of the preparation is 0.1-0.5% of the total feed mass; And / or, the pig is a fattening pig.

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