Traditional Chinese medicine extract for relieving weak piglets and preparation method thereof

Abstract

The application discloses a traditional Chinese medicine extract for relieving weak piglets and a preparation method thereof, and particularly relates to the technical field of veterinary traditional Chinese medicines, which is composed of 15-25 parts of total ginsenosides of acanthopanax, 8-15 parts of astragaloside IV, 3-8 parts of atractylodes lactone III, 5-12 parts of notoginseng glycoside, 20-35 parts of pachyman, 10-20 parts of total organic acid of hawthorn and 5-10 parts of fermented short-chain fatty acid. The preparation method is as follows: medicinal materials are subjected to complex enzymolysis, liquid submerged fermentation of lactobacillus plantarum, macroporous adsorption resin column chromatography separation of effective parts, and then quantitative compounding and drying to obtain the extract. The extract has high effective component dissolution rate, controllable quality, can significantly improve the daily weight gain of weak piglets, reduce the feed-weight ratio and diarrhea rate, repair the intestinal mucosal barrier, and enhance the immunity, and the total effective rate of the extract for relieving weak piglets of various types reaches more than 93%, so that the extract can be applied as a veterinary drug or a feed additive.

Description

Technical Field

[0001] This invention relates to the field of veterinary traditional Chinese medicine technology, and more specifically, to a traditional Chinese medicine extract for relieving stiffness and weakness in piglets and its preparation method. Background Technology

[0002] Stunted piglets, also known as stunted pigs or legless pigs, are a common growth disorder in pig production. Clinical manifestations include lethargy, rough coat, low feed intake, and stunted growth, with piglets weighing only 50%–70% of healthy piglets in their litter, and a significantly elevated feed conversion ratio. Depending on the cause, stunted piglets can be classified into four types: fetal stunted piglets, milk stunted piglets, feed stunted piglets, and disease-related stunted piglets. Disease-related stunted piglets, occurring after diarrhea, are the most common, accounting for over 50% of all stunted piglets. The incidence rate in large-scale pig farms is typically 10%–20%, but can reach over 30% in poorly managed farms. Affected piglets experience a 2–3 month extension in their growth cycle, a decrease in feed efficiency of over 30%, and in severe cases, they may become old and weak or even die, causing significant economic losses to the pig farming industry.

[0003] Modern research indicates that the core pathological mechanism of stunted growth in piglets is a vicious cycle of intestinal mucosal barrier damage, immune dysfunction, and growth axis inhibition. After weaning or diarrhea, piglets experience villus atrophy, crypt hyperplasia, decreased expression of tight junction proteins, and increased intestinal permeability. Simultaneously, immune organs are underdeveloped, immunoglobulin levels are reduced, and resistance to pathogens decreases. This leads to suppression of the growth hormone-insulin-like growth factor axis, a significant decrease in serum IGF-1 levels, and ultimately, growth arrest.

[0004] Currently, clinical treatment for stunted growth in piglets mainly employs two methods: Western medicine and traditional Chinese medicine. Western medicine often uses antibiotics, trace element preparations, vitamins, and growth hormones. While these methods have some effect, they suffer from problems such as drug resistance, drug residues, and high costs, and are unlikely to fundamentally repair intestinal mucosal damage and rebuild immune function. Traditional Chinese veterinary medicine believes that stunted growth in piglets is mostly caused by spleen and stomach weakness and insufficient qi and blood. Treatment focuses on strengthening the spleen and stomach, replenishing qi and nourishing blood, and commonly uses Chinese herbal powders or decoctions.

[0005] The prior art discloses a variety of traditional Chinese medicine compound formulas for treating stunted pigs, such as a raw herb powder composed of Rosa laevigata, Cuscuta chinensis, Cornus officinalis, Alpinia oxyphylla, Codonopsis pilosula, Astragalus membranaceus, Atractylodes macrocephala, Polygala tenuifolia, Acanthopanax senticosus, and Glycyrrhiza uralensis, which has achieved certain clinical efficacy.

[0006] However, existing traditional Chinese medicine preparations generally have the following shortcomings: First, most of them use raw medicinal materials directly pulverized and added into the medicine, without extraction and separation of effective components, resulting in low dissolution rate and poor bioavailability; second, they lack quantitative control of effective parts, resulting in unstable product quality and large batch-to-batch differences; third, the formulations are mostly based on strengthening the spleen and improving appetite, and lack multi-target synergistic effects on intestinal mucosal barrier repair, immune function reconstruction and growth axis restart; fourth, they have not introduced microbial fermentation technology, and have failed to utilize the nutritional and repair effects of short-chain fatty acids and other metabolites produced by fermentation on the intestine.

[0007] Therefore, developing a traditional Chinese medicine extract for treating piglet stunting and weakness with clearly defined effective components, controllable quality, significant efficacy, and a clear mechanism of action has important clinical significance and application value. Summary of the Invention

[0008] To achieve the above objectives, the present invention provides the following technical solution: A traditional Chinese medicine extract for relieving stunted growth in piglets, by weight, its active ingredients are mainly composed of the following effective components: The ingredients include 15-25 parts of total glycosides of Acanthopanax senticosus, 8-15 parts of astragaloside A, 3-8 parts of atractylodes lactone III, 5-12 parts of codonopsis glycosides, 20-35 parts of poria cocos polysaccharide, 10-20 parts of total organic acids from hawthorn, and 5-10 parts of short-chain fatty acids produced by fermentation. The short-chain fatty acid is a mixture of lactic acid and acetic acid in a mass ratio of 3 to 5:1.

[0009] Preferably, the active ingredient, by weight, is mainly composed of the following effective fractions: The contents included 18-22 parts of total glycosides of Acanthopanax senticosus, 10-13 parts of astragaloside A, 4-6 parts of atractylodes lactone III, 7-10 parts of codonopsis glycosides, 25-30 parts of poria cocos polysaccharides, 13-17 parts of total organic acids from hawthorn, and 6-8 parts of short-chain fatty acids produced by fermentation.

[0010] Preferably, the total glycosides of Acanthopanax senticosus are a mixture of Acanthopanax senticosus glycoside B and Acanthopanax senticosus glycoside E, wherein the mass fraction of Acanthopanax senticosus glycoside E is not less than 45%; The mass fraction of citric acid in the total organic acids of hawthorn shall not be less than 50%.

[0011] A method for preparing a traditional Chinese medicine extract to alleviate stunted growth in piglets, comprising the following steps: S1. Pretreatment of medicinal materials: Weigh 25-35 parts of Acanthopanax senticosus, 20-30 parts of Astragalus membranaceus, 10-18 parts of Atractylodes macrocephala, 10-18 parts of Codonopsis pilosula, 15-25 parts of Poria cocos, and 10-20 parts of Crataegus pinnatifida according to the weight ratio of raw herbs, mix them, and coarsely grind them to 20-40 mesh to obtain mixed powder of medicinal materials. S2. Compound enzymatic hydrolysis: Add acetate-sodium acetate buffer solution with pH 4.5-5.5 to the mixed powder of medicinal materials at a ratio of 8-12 mL per gram of medicinal materials, soak for 30-60 min, then add a compound enzyme accounting for 0.3%-0.8% of the total mass of medicinal materials. The compound enzyme is composed of cellulase, pectinase and neutral protease in an enzyme activity ratio of 2-3:1-2:1. Hydrolyze at a constant temperature of 45-55℃ for 2-4 h to obtain the enzymatic hydrolysate. S3. Sterilization and cooling: Boil the enzymatic hydrolysate for 10-15 minutes to inactivate the enzymes, and then cool it to 35-38°C. S4. Liquid deep fermentation: Inoculate the cooled enzymatic hydrolysate with activated Lactobacillus plantarum culture. The inoculum size is 2%–5% by volume, and the initial bacterial count is not less than 1×10⁻⁶. 8 The concentration of CFU / mL was sealed and then anaerobic fermented at 35–37°C for 48–72 h to obtain the fermentation broth. S5. Separation, Purification, and Standardization: Centrifuge the fermentation broth to obtain the supernatant, filter it through a 0.22 μm microfiltration membrane, and then pass the filtrate onto a macroporous adsorption resin column. First, elute with water and collect the water wash containing short-chain fatty acids. Then, elute with a gradient of 30%–90% ethanol, collecting the alcohol eluents containing total Acanthopanax senticosus glycosides, astragaloside A, atractylodes lactone III, codonopsis glycosides, poria cocos polysaccharides, and total organic acids from hawthorn. Determine the content of effective components in each fraction, combine the alcohol eluents with the water wash, and make the resulting extract contain 15–25 parts by weight of total Acanthopanax senticosus glycosides, 8–15 parts by weight of astragaloside A, and atractylodes lactone III. The following ingredients are added: 3-8 parts by weight, 5-12 parts by weight of codonopsis glycosides, 20-35 parts by weight of poria cocos polysaccharide, 10-20 parts by weight of total organic acids from hawthorn, and 5-10 parts by weight of short-chain fatty acids. The mixture is concentrated under reduced pressure to a relative density of 1.10-1.15 at 60°C, and then spray-dried or freeze-dried to obtain the final product.

[0012] Preferably, in step S2, the cellulase activity is not less than 40,000 U / g, the pectinase activity is not less than 30,000 U / g, and the neutral protease activity is not less than 100,000 U / g; the enzymatic hydrolysis conditions are: temperature 50℃, pH 5.0, time 3h, and the total amount of the compound enzyme added is 0.5% of the total mass of the medicinal materials.

[0013] Preferably, in step S4, the fermentation conditions are: temperature 37℃, inoculum size 3% by volume, and fermentation time 60h; at the end of fermentation, the pH of the fermentation broth is 3.8–4.2, and the viable count of *Lactobacillus plantarum* is not less than 1×10⁻⁶. 9 CFU / mL.

[0014] Preferably, in step S5, the macroporous adsorption resin is an AB-8 type or a D101 type non-polar macroporous adsorption resin; short-chain fatty acids, poria cocos polysaccharides, and total organic acids from hawthorn are collected in the water eluent; the ethanol gradient elution is as follows: 30% ethanol elution to collect the codonopsis glycoside fraction, 50% ethanol elution to collect the total glycoside fraction from Acanthopanax senticosus, 70% ethanol elution to collect the astragaloside A and atractylodes lactone III fractions, and the 90% ethanol eluent is discarded; the fractions are combined after their content is detected by high performance liquid chromatography.

[0015] This invention also provides the application of traditional Chinese medicine extracts in the preparation of veterinary drugs or feed additives to alleviate stunted growth in piglets.

[0016] Preferably, the stunted growth of piglets includes fetal stunted growth, milk stunted growth, food stunted growth, and disease stunted growth, especially growth retardation caused by intestinal villus atrophy and poor development of immune organs following diarrhea; when the traditional Chinese medicine extract is used as a feed additive, the amount added to the basic diet of piglets is 0.2% to 0.5% by weight, and the continuous feeding for 10 to 14 days constitutes one course of treatment.

[0017] A veterinary drug composition for relieving stunted growth in piglets comprises an effective amount of the above-mentioned traditional Chinese medicine extract, and a veterinary pharmaceutically acceptable carrier or excipient.

[0018] The technical effects and advantages of this invention are as follows: 1. This invention employs a combined enzymatic hydrolysis and Lactobacillus plantarum liquid deep fermentation technology, combined with macroporous adsorption resin column chromatography separation and standardized formulation process, significantly improving the dissolution rate and extraction efficiency of effective components in traditional Chinese medicine. Experiments show that compared with enzymatic hydrolysis or fermentation alone, the process of this invention achieves a total glycoside dissolution rate of 75.2% for Acanthopanax senticosus, 68.9% for astragaloside A, and 70.1% for atractylodes lactone III, demonstrating a significant synergistic effect between enzymatic hydrolysis and microbial fermentation. Simultaneously, column chromatography separation and quantitative formulation ensure precise control of the content of each effective component, good batch-to-batch stability, and suitability for large-scale industrial production. 2. The herbal extract of this invention has a significant therapeutic effect on stunted growth in piglets, significantly improving their growth performance and reducing diarrhea rates. Clinical trials show that adding 0.3% of the extract of this invention to the basal diet for 10 consecutive days resulted in an average daily weight gain of 426g / d for stunted piglets, which is 158% higher than the control group and 52.7% higher than the existing technology group; the feed conversion ratio decreased to 1.78, the diarrhea rate decreased to 3.3%, and the total effective rate reached 93.3%; the serum insulin-like growth factor 1 (IGF-1) level reached 156.2ng / mL, which is 24.7% higher than the existing technology group, effectively restarting the growth axis of piglets. 3. This invention works synergistically through multiple pathways, including repairing the intestinal mucosal barrier, enhancing the body's immune function, and restarting the growth axis, showing good therapeutic effects on different types of stunted growth in piglets. Experiments show that the extract of this invention can increase jejunal villus height by 21.0%, the villus-gland ratio by 26.4%, the expression of tight junction protein occludin by 36.6%, the spleen index by 22.8%, and serum IgG by 27.9% compared to existing technologies. The overall effective rate for four types of stunted piglets—fetal stunted growth, milk stunted growth, food stunted growth, and disease stunted growth—reaches 95%, with a particularly high effective rate of 90% for secondary disease stunted growth following diarrhea, which is clinically common and difficult to treat. The extract of this invention is a standardized composition of effective components, with controllable quality and convenient use, and can be used as a feed additive or veterinary drug. Attached Figure Description

[0019] Figure 1 This is a flowchart of the preparation method of the present invention. Detailed Implementation

[0020] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

[0021] The *Lactobacillus plantarum* CGMCC 1.557 used in this invention was purchased from the China General Microbiological Culture Collection Center. Cellulase activity (40,000 U / g), pectinase activity (30,000 U / g), and neutral protease activity (100,000 U / g) were all purchased from Nanning Pangbo Biotechnology Co., Ltd. Acanthopanax senticosin B, Acanthopanax senticosin E, Astragaloside A, Atractylodes lactone III, Codonopsis pilosula glycoside, Poria cocos polysaccharide, and citric acid reference standards were all purchased from the China National Institutes for Food and Drug Control.

[0022] Example 1: As attached Figure 1 As shown in the figure, this embodiment provides a traditional Chinese medicine extract for relieving stunted growth in piglets. The preparation method of this traditional Chinese medicine extract is as follows: S1. Pretreatment of medicinal materials Weigh out the raw herbs according to the following weight ratios: 30 parts Acanthopanax senticosus, 25 parts Astragalus membranaceus, 14 parts Atractylodes macrocephala, 14 parts Codonopsis pilosula, 20 parts Poria cocos, and 15 parts Crataegus pinnatifida. Coarsely pulverize the above herbs using a universal pulverizer, and pass them through a 30-mesh sieve to obtain a mixed powder.

[0023] S2, compound enzymatic hydrolysis Take 10 kg of mixed medicinal powder and add 100 L of pH 5.0 acetate-sodium acetate buffer solution. The volume of this buffer solution is 10 times the weight-to-volume ratio of the medicinal materials (g / mL). Soak for 45 min to allow the medicinal materials to fully absorb water and swell. Add 50 g of a compound enzyme, including 25 g of cellulase, 15 g of pectinase, and 10 g of neutral protease, with an enzyme activity ratio of 2.5:1.5:1. Stir well and incubate at 50℃ for 3 h, stirring every 30 min to obtain the enzymatic hydrolysate.

[0024] S3, Sterilization and Cooling Heat the enzymatic hydrolysate to boiling, hold for 12 minutes to inactivate the enzyme, and then rapidly cool to 37°C.

[0025] S4, Liquid Deep Fermentation Lactobacillus plantarum CGMCC 1.557 slant culture was inoculated into MRS liquid medium and incubated statically at 37℃ for 24 h. After microscopic examination showed no contamination, the culture was used as the seed culture, with a viable count of 1.5 × 10⁻⁶. 9 CFU / mL. The seed culture was inoculated into the cooled enzymatic hydrolysate at a 3% (v / v) inoculum ratio, resulting in an initial bacterial count of 1.5 × 10⁻⁶. 8 CFU / mL. The fermentation broth was obtained by anaerobic fermentation in a sealed fermenter at 37°C for 60 hours. At the end of fermentation, the pH was measured to be 4.0, and the viable count of *Lactobacillus plantarum* was 1.6 × 10⁻⁶. 9 CFU / mL.

[0026] S5. Separation, Purification and Standardization The fermentation broth was centrifuged at 8000 rpm for 15 min, and the supernatant was collected. The supernatant was then sterilized and clarified through a 0.22 μm microfiltration membrane to obtain a clear filtrate. The clear filtrate was loaded onto a pre-treated D101 macroporous adsorption resin column with a column diameter-to-height ratio of 1:8, a loading flow rate of 2 BV / h, and a loading volume of 3 BV. After loading, the column was eluted with 4 BV of deionized water at a flow rate of 3 BV / h, and the washings were collected. This wash contained short-chain fatty acids, poria cocos polysaccharides, and total organic acids from hawthorn. A gradient elution was then performed sequentially with 4 BV of 30% ethanol, 4 BV of 50% ethanol, 6 BV of 70% ethanol, and 4 BV of 90% ethanol at a flow rate of 2 BV / h. The 30%, 50%, 70%, and 90% ethanol eluates were collected, respectively. Testing revealed that the 30% ethanol eluent mainly contained codonopsis glycosides, the 50% ethanol eluent mainly contained total glycosides of Acanthopanax senticosus, the 70% ethanol eluent mainly contained astragaloside A and atractylodes lactone III, and the 90% ethanol eluent contained a small amount of lipid-soluble impurities, which were discarded. The ethanol in each of the above effective fractions was recovered under reduced pressure and concentrated, and the content of effective components in each fraction was determined. According to the weight proportions described in claim 1, the concentrated solutions of each fraction were accurately measured and combined with the washing solution, mixed thoroughly, so that the weight proportions of each effective component in the total solids conformed to the range of 15-25 parts of total glycosides of Acanthopanax senticosus, 8-15 parts of astragaloside A, 3-8 parts of atractylodes lactone III, 5-12 parts of codonopsis glycosides, 20-35 parts of Poria cocos polysaccharides, 10-20 parts of total organic acids from hawthorn, and 5-10 parts of short-chain fatty acids. The combined medicinal liquid was concentrated to a relative density of 1.12 at 60℃ and -0.08MPa, and then spray-dried at an inlet air temperature of 160℃ and an outlet air temperature of 80℃ to obtain approximately 2.1 kg of brownish-yellow powdery Chinese herbal extract, with a yield of approximately 21%.

[0027] The content determination method is as follows. Total glycosides of Acanthopanax senticosus were determined by HPLC. The chromatographic conditions were: Agilent C18 column, mobile phase: acetonitrile-0.1% phosphoric acid aqueous solution gradient elution, detection wavelength: 220 nm, column temperature: 30℃, flow rate: 1.0 mL / min. Accurately weigh appropriate amounts of Acanthopanax senticosus glycoside B and Acanthopanax senticosus glycoside E reference standards, and add methanol to prepare a mixed reference solution containing 0.2 mg of each per mL. For the test solution, accurately weigh approximately 0.5 g of the sample, place it in a 25 mL volumetric flask, add 20 mL of methanol, sonicate for 30 min, cool, add methanol to the mark, shake well, filter, and collect the filtrate. Accurately inject 10 μL each of the reference solution and the test solution into the HPLC system for determination, and calculate the content based on peak area using the external standard method.

[0028] Astragaloside A was determined by HPLC-ELSD. The chromatographic conditions were: Agilent C18 column, mobile phase acetonitrile-water 32:68, flow rate 1.0 mL / min, column temperature 30℃, drift tube temperature 105℃, and carrier gas flow rate 2.5 L / min. Atractylodes lactone III was determined by HPLC, mobile phase methanol-water 70:30, and detection wavelength 220 nm. Codonopsis pilosula glycosides were determined by HPLC, mobile phase acetonitrile-0.1% phosphoric acid aqueous solution 25:75, and detection wavelength 267 nm. Poria cocos polysaccharides were determined by the phenol-sulfuric acid method, with anhydrous glucose as a control. Short-chain fatty acids were determined by HPLC, chromatographic conditions were: Aminex HPX-87H column, mobile phase 5 mmol / L sulfuric acid solution, flow rate 0.6 mL / min, column temperature 50℃, and detection wavelength 210 nm.

[0029] Tests showed that each gram of extract contained 20.3 mg of total glycosides of Acanthopanax senticosus, including 9.7 mg of Acanthopanax senticosus glycoside E, which accounted for 47.8% of the total glycosides of Acanthopanax senticosus; 11.5 mg of astragaloside A, 4.8 mg of atractylodes lactone III, 8.2 mg of codonopsis glycoside, and 27.6 mg of Poria cocos polysaccharide; 15.1 mg of total organic acids from hawthorn, including 8.6 mg of citric acid, which accounted for 57.0% of the total organic acids from hawthorn; and 6.5 mg / g of short-chain fatty acids, including 5.2 mg of lactic acid and 1.3 mg of acetic acid, with a lactic acid to acetic acid mass ratio of 4:1.

[0030] Example 2: This embodiment provides a traditional Chinese medicine extract for alleviating stunted growth in piglets, which differs from Embodiment 1 in that: S1. Pretreatment of medicinal materials Weigh out the raw herbs according to the following weight ratios: 25 parts Acanthopanax senticosus, 20 parts Astragalus membranaceus, 10 parts Atractylodes macrocephala, 10 parts Codonopsis pilosula, 15 parts Poria cocos, and 10 parts Crataegus pinnatifida. After coarsely pulverizing the herbs, pass them through a 20-mesh sieve.

[0031] S2, compound enzymatic hydrolysis Add 80 L of pH 4.5 acetate-sodium acetate buffer (8 g / mL weight-to-volume ratio) and soak for 30 min. Add 30 g of a complex enzyme, including 18 g of cellulase, 8 g of pectinase, and 4 g of neutral protease, with an enzyme activity ratio of 3:2:1. Incubate at 45℃ for 4 h.

[0032] S3, Sterilization and Cooling Boil for 10 minutes to inactivate the enzyme, then cool to 35°C.

[0033] S4, Liquid Deep Fermentation Inoculate the seed culture at a 2% volume ratio, with an initial bacterial count of 1.0 × 10⁻⁶. 8CFU / mL. Anaerobic fermentation was carried out at 35℃ for 72 h. At the end of fermentation, the pH was measured to be 3.8, and the viable count of *Lactobacillus plantarum* was 1.2 × 10⁻⁶. 9 CFU / mL.

[0034] S5. Separation, Purification and Standardization The separation, purification and standardization steps were the same as in Example 1. After combining the drug solutions, the concentration was increased to a relative density of 1.10, and then freeze-dried.

[0035] The extract contained 15.2 mg of total glycosides of Acanthopanax senticosus per gram, including 6.9 mg of Acanthopanax senticosus glycoside E, which accounted for 45.4% of the total glycosides of Acanthopanax senticosus; 8.3 mg of astragaloside A, 3.1 mg of atractylodes lactone III, 5.4 mg of codonopsis glycoside, and 20.7 mg of Poria cocos polysaccharide; 10.3 mg of total organic acids of hawthorn, including 5.2 mg of citric acid, which accounted for 50.5% of the total organic acids of hawthorn; and 5.1 mg / g of short-chain fatty acids, including 3.8 mg of lactic acid and 1.3 mg of acetic acid, with a lactic acid to acetic acid mass ratio of 2.9:1.

[0036] Example 3: This embodiment provides a traditional Chinese medicine extract for alleviating stunted growth in piglets, which is basically the same as that in Example 1, except that: S1. Pretreatment of medicinal materials Weigh out the raw herbs according to the following weight ratios: 35 parts Acanthopanax senticosus, 30 parts Astragalus membranaceus, 18 parts Atractylodes macrocephala, 18 parts Codonopsis pilosula, 25 parts Poria cocos, and 20 parts Crataegus pinnatifida. After coarsely pulverizing the herbs, pass them through a 40-mesh sieve.

[0037] S2, compound enzymatic hydrolysis Add 120 L of pH 5.5 acetate-sodium acetate buffer (12 times the weight-to-volume ratio of g / mL) and soak for 60 min. Add 80 g of a complex enzyme, including 36 g of cellulase, 28 g of pectinase, and 16 g of neutral protease, with an enzyme activity ratio of 2:1.5:1. Incubate at 55℃ for 2 h.

[0038] S3, Sterilization and Cooling Boil for 15 minutes to inactivate the enzyme, then cool to 38°C.

[0039] S4, Liquid Deep Fermentation Inoculate the seed culture at a 5% volume ratio, with an initial bacterial count of 2.0 × 10⁻⁶. 8 CFU / mL. Anaerobic fermentation was carried out at 37℃ for 48 h. At the end of fermentation, the pH was measured to be 4.2, and the viable count of *Lactobacillus plantarum* was 2.1 × 10⁻⁶. 9 CFU / mL.

[0040] S5. Separation, Purification and Standardization The separation, purification, and standardization steps were the same as in Example 1. After combining the drug solutions, the concentration was increased to a relative density of 1.15, and then spray-dried.

[0041] The extract contained 24.7 mg of total glycosides of Acanthopanax senticosus per gram, including 12.1 mg of Acanthopanax senticosus glycoside E, which accounted for 49.0% of the total glycosides of Acanthopanax senticosus; 14.6 mg of astragaloside A, 7.8 mg of atractylodes lactone III, 11.5 mg of codonopsis glycoside, and 34.2 mg of Poria cocos polysaccharide; 19.6 mg of total organic acids from hawthorn, including 11.2 mg of citric acid, which accounted for 57.1% of the total organic acids from hawthorn; and 9.8 mg / g of short-chain fatty acids, including 8.1 mg of lactic acid and 1.7 mg of acetic acid, with a lactic acid to acetic acid mass ratio of 4.8:1.

[0042] Comparative Example 1: The control drug was prepared according to the method disclosed in the prior art. The following ingredients were weighed by weight percentage: 12% Rosa laevigata, 8% Cuscuta chinensis, 10% Cornus officinalis, 12% Alpinia oxyphylla, 12% Codonopsis pilosula, 10% Astragalus membranaceus, 13% Atractylodes macrocephala, 12% Polygala tenuifolia, 8% Acanthopanax senticosus, and 3% Glycyrrhiza uralensis. The above-mentioned medicinal materials were coarsely pulverized, passed through a 5mm sieve, dried to 10% moisture content, cooled, finely pulverized, passed through a 1mm sieve, and mixed evenly to obtain the raw herb powder.

[0043] Comparative Example 2: 10 kg of raw medicinal materials were weighed according to the formula in Example 1, coarsely pulverized and passed through a 30-mesh sieve, and soaked in 100 L of pH 5.0 acetate-sodium acetate buffer for 45 min. Without enzymatic hydrolysis, the mixture was directly boiled for 12 min and cooled to 37°C. Lactobacillus plantarum CGMCC 1.557 seed culture was inoculated at a 3% (v / v) ratio, and anaerobic fermentation was carried out at 37°C for 60 h. The fermentation broth was centrifuged, microfiltered, and purified by separation using a D101 macroporous adsorption resin column. Elution with 70% ethanol, concentration, and drying yielded the unenzymatically hydrolyzed direct fermentation control extract.

[0044] Comparative Example 3: According to the crude drug formulation in Example 1, 10 kg of medicinal materials were weighed, coarsely pulverized, and passed through a 30-mesh sieve. 100 L of pH 5.0 acetate-sodium acetate buffer was added, and the mixture was soaked for 45 min. 50 g of a compound enzyme was added, and the mixture was enzymatically hydrolyzed at 50°C for 3 h. The enzyme was inactivated by boiling for 12 min. After cooling, without inoculating with *Lactobacillus plantarum* or performing a fermentation step, the mixture was directly centrifuged, microfiltered, and purified using a D101 macroporous adsorption resin column. Elution with 70% ethanol was performed, and the extract was concentrated and dried to obtain the control extract obtained only from enzymatic hydrolysis without fermentation.

[0045] Experimental Example 1: Samples from Example 1, Comparative Example 2, and Comparative Example 3 were taken respectively, and the contents of total glycosides of Acanthopanax senticosus, astragaloside A, and atractylodes lactone III were determined according to the content determination method described in Example 1. The dissolution rate was calculated based on the theoretical content in the crude drug. The results are shown in Table 1.

[0046] Table 1: Comparison of dissolution rates of effective components under different processes As shown in Table 1, the combined enzymatic hydrolysis-fermentation process in Example 1 significantly increased the dissolution rate of each active ingredient compared to enzymatic hydrolysis or fermentation alone. Compared to direct fermentation without enzymatic hydrolysis, the dissolution rate of total glycosides from Acanthopanax senticosus increased by 1.2 times, astragaloside A by 74.4%, and atractylodes lactone III by 1.6 times. Compared to enzymatic hydrolysis without fermentation, the dissolution rate of total glycosides from Acanthopanax senticosus increased by 26.0%, astragaloside A by 28.1%, and atractylodes lactone III by 44.2%. These results demonstrate a significant synergistic effect between combined enzymatic hydrolysis and microbial fermentation.

[0047] Experimental Example 2: The experiment was conducted at a large-scale pig farm in a certain region. Sixty 45-day-old weaned piglets clinically diagnosed as stunted pigs were selected. Inclusion criteria included a body weight of only 50%–65% of the average weight of healthy piglets in their litter, rough and disheveled coat, lethargy, low feed intake, growth retardation for more than 7 days, and some having a history of diarrhea. The experimental pigs were randomly divided into 4 groups of 15 piglets each, based on litter, body weight, and sex. Statistical analysis showed no significant difference in initial body weight among the groups.

[0048] The blank control group was fed a basal diet. The existing technology group was fed a basal diet supplemented with 1% of the raw herbal powder from Example 1, which is the recommended dosage in the existing technology. The low-dose group of this invention was fed a basal diet supplemented with 0.2% of the extract from Example 1. The high-dose group of this invention was fed a basal diet supplemented with 0.3% of the extract from Example 1. All experimental pigs were housed in the same nursery, with free access to feed and water, and were immunized according to the standard immunization program. The experimental period was 10 days.

[0049] Feed intake was recorded daily during the experiment, and diarrhea was observed and the number of pigs experiencing diarrhea was recorded. At the beginning and end of the experiment, each pig was weighed on an empty stomach in the morning to calculate the average daily weight gain and feed conversion ratio. On the last day of the experiment, blood was collected from the anterior vena cava of 10 pigs in each group, serum was separated, and serum IGF-1 levels were measured using a porcine insulin-like growth factor 1 ELISA kit, serum IgG levels were measured using a porcine immunoglobulin G ELISA kit, and serum TNF-α levels were measured using a porcine tumor necrosis factor α ELISA kit.

[0050] The criteria for determining the therapeutic effect are as follows: A marked effect is defined as a final weight reaching 80% or more of the average weight of healthy piglets from the same litter, with good mental state, smooth coat, and normal feed intake. An effective effect is defined as a final weight reaching 60%–80% of the average weight of healthy piglets from the same litter, with significant improvement in mental state and appetite. An ineffective effect is defined as a final weight below 60% of the average weight of healthy piglets from the same litter, with significantly stunted growth. The total effective rate is the sum of the marked effect rate and the effective rate.

[0051] The test results are shown in Table 2.

[0052] Table 2: Comparison of growth performance and therapeutic effects in each group of stunted pigs As shown in Table 2, the average daily weight gain in the 0.3% dose group of this invention reached 426 g / d, which is 158% higher than that of the blank control group and 52.7% higher than that of the prior art group. The feed conversion ratio decreased to 1.78, and the diarrhea rate decreased to 3.3%. The total effective rate reached 93.3%, which is 13.3 percentage points higher than that of the prior art group. The serum IGF-1 content reached 156.2 ng / mL, which is 89.1% higher than that of the blank control group and 24.7% higher than that of the prior art group.

[0053] At the end of the experiment, four pigs from each group were randomly selected and euthanized by exsanguination. The abdominal cavity was immediately dissected, and approximately 2 cm of tissue from the mid-jejunum was harvested. This tissue was fixed in 4% paraformaldehyde, routinely embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), and the intestinal mucosal morphology was observed under a light microscope. Villus height and crypt depth were measured, and the villus-to-gland ratio was calculated. The expression level of the intestinal mucosal tight junction protein occludin was detected using immunohistochemical SP method, and expressed as the average integrated optical density value. The spleen was harvested, weighed, and the spleen index was calculated.

[0054] The results of intestinal morphology and immune index measurements are shown in Table 3.

[0055] Table 3: Comparison of intestinal morphology and immune indicators among groups As shown in Table 3, the 0.3% group of this invention showed significantly higher jejunal villus height, villus-to-gland ratio, occludin expression level, spleen index, and serum IgG content compared to the prior art group. Villus height increased by 21.0%, villus-to-gland ratio by 26.4%, occludin expression by 36.6%, spleen index by 22.8%, and serum IgG by 27.9% compared to the prior art group. These results demonstrate that this invention can indeed promote intestinal mucosal structure repair, enhance intestinal barrier function, and improve the body's immunity.

[0056] Experiment Example 3: Treatment Effects on Different Types of Runts To verify the therapeutic effect of this invention on different types of stunted pigs, 10 pigs each of fetal stunted pigs, milk stunted pigs, food stunted pigs, and disease stunted pigs were selected, for a total of 40 pigs. Fetal stunted pigs were piglets with a birth weight of less than 0.8 kg and slow growth. Milk stunted pigs were piglets with slow growth due to insufficient milk production during lactation. Food stunted pigs were piglets with slow growth due to poor feed quality or insufficient feed intake after weaning. Disease stunted pigs were piglets whose growth had stopped after recovery from diarrhea or respiratory diseases. All pigs were fed a basal diet containing 0.3% of the extract from Example 1 for 10 consecutive days, and the therapeutic effect was evaluated using average daily weight gain and total effective rate as indicators.

[0057] The results showed that the average daily weight gain in the fetal stunted pig group was 387g / d, with a total effective rate of 90%. The average daily weight gain in the milk stunted pig group was 442g / d, with a total effective rate of 100%. The average daily weight gain in the feed stunted pig group was 468g / d, with a total effective rate of 100%. The average daily weight gain in the diseased stunted pig group was 395g / d, with a total effective rate of 90%. The total effective rate of the four groups was 95%. The results demonstrate that this invention has a good therapeutic effect on various types of stunted pigs, especially achieving an excellent 90% effect on the most common and difficult-to-treat diseased stunted pigs.

[0058] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A traditional Chinese medicine extract for relieving stunted growth in piglets, characterized in that: Based on parts by weight, its active ingredients mainly consist of the following effective fractions: The ingredients include 15-25 parts of total glycosides of Acanthopanax senticosus, 8-15 parts of astragaloside A, 3-8 parts of atractylodes lactone III, 5-12 parts of codonopsis glycosides, 20-35 parts of poria cocos polysaccharide, 10-20 parts of total organic acids from hawthorn, and 5-10 parts of short-chain fatty acids produced by fermentation. The short-chain fatty acid is a mixture of lactic acid and acetic acid in a mass ratio of 3 to 5:

1.

2. The traditional Chinese medicine extract for relieving stunted growth in piglets according to claim 1, characterized in that: Based on parts by weight, its active ingredients mainly consist of the following effective fractions: The contents included 18-22 parts of total glycosides of Acanthopanax senticosus, 10-13 parts of astragaloside A, 4-6 parts of atractylodes lactone III, 7-10 parts of codonopsis glycosides, 25-30 parts of poria cocos polysaccharides, 13-17 parts of total organic acids from hawthorn, and 6-8 parts of short-chain fatty acids produced by fermentation.

3. The traditional Chinese medicine extract for relieving stunted growth in piglets according to claim 1, characterized in that: The total glycosides of Acanthopanax senticosus are a mixture of Acanthopanax senticosus glycoside B and Acanthopanax senticosus glycoside E, wherein the mass fraction of Acanthopanax senticosus glycoside E is not less than 45%. The mass fraction of citric acid in the total organic acids of hawthorn shall not be less than 50%.

4. A method for preparing a traditional Chinese medicine extract for relieving stunted growth in piglets, used to prepare the traditional Chinese medicine extract according to any one of claims 1 to 3, characterized in that: Includes the following steps: S1. Pretreatment of medicinal materials: Weigh 25-35 parts of Acanthopanax senticosus, 20-30 parts of Astragalus membranaceus, 10-18 parts of Atractylodes macrocephala, 10-18 parts of Codonopsis pilosula, 15-25 parts of Poria cocos, and 10-20 parts of Crataegus pinnatifida according to the weight ratio of raw herbs, mix them, and coarsely grind them to 20-40 mesh to obtain mixed powder of medicinal materials. S2. Compound enzymatic hydrolysis: Add acetate-sodium acetate buffer solution with pH 4.5-5.5 to the mixed powder of medicinal materials at a ratio of 8-12 mL per gram of medicinal materials, soak for 30-60 min, then add a compound enzyme accounting for 0.3%-0.8% of the total mass of medicinal materials. The compound enzyme is composed of cellulase, pectinase and neutral protease in an enzyme activity ratio of 2-3:1-2:

1. Hydrolyze at a constant temperature of 45-55℃ for 2-4 h to obtain the enzymatic hydrolysate. S3. Sterilization and cooling: Boil the enzymatic hydrolysate for 10-15 minutes to inactivate the enzymes, and then cool it to 35-38°C. S4. Liquid deep fermentation: Inoculate the cooled enzymatic hydrolysate with activated Lactobacillus plantarum culture. The inoculum size is 2%–5% by volume, and the initial bacterial count is not less than 1×10⁻⁶. 8 The concentration of CFU / mL was sealed and then anaerobic fermented at 35–37°C for 48–72 h to obtain the fermentation broth. S5. Separation, Purification, and Standardization: Centrifuge the fermentation broth to obtain the supernatant, filter it through a 0.22 μm microfiltration membrane, and then pass the filtrate onto a macroporous adsorption resin column. First, elute with water and collect the water wash containing short-chain fatty acids. Then, elute with a gradient of 30%–90% ethanol, collecting the alcohol eluents containing total Acanthopanax senticosus glycosides, astragaloside A, atractylodes lactone III, codonopsis glycosides, poria cocos polysaccharides, and total organic acids from hawthorn. Determine the content of effective components in each fraction, combine the alcohol eluents with the water wash, and make the resulting extract contain 15–25 parts by weight of total Acanthopanax senticosus glycosides, 8–15 parts by weight of astragaloside A, and atractylodes lactone III. The following ingredients are added: 3-8 parts by weight, 5-12 parts by weight of codonopsis glycosides, 20-35 parts by weight of poria cocos polysaccharide, 10-20 parts by weight of total organic acids from hawthorn, and 5-10 parts by weight of short-chain fatty acids. The mixture is concentrated under reduced pressure to a relative density of 1.10-1.15 at 60°C, and then spray-dried or freeze-dried to obtain the final product.

5. The preparation method according to claim 4, characterized in that: In step S2, the cellulase activity is not less than 40,000 U / g, the pectinase activity is not less than 30,000 U / g, and the neutral protease activity is not less than 100,000 U / g; the enzymatic hydrolysis conditions are: temperature 50℃, pH 5.0, time 3h, and the total amount of the compound enzyme added is 0.5% of the total mass of the medicinal materials.

6. The preparation method according to claim 5, characterized in that: In step S4, the fermentation conditions are: temperature 37℃, inoculum size 3% by volume, and fermentation time 60h; at the end of fermentation, the pH of the fermentation broth is 3.8–4.2, and the viable count of *Lactobacillus plantarum* is not less than 1×10⁻⁶. 9 CFU / mL.

7. The preparation method according to claim 4, characterized in that: In step S5, the macroporous adsorption resin is AB-8 or D101 type nonpolar macroporous adsorption resin; short-chain fatty acids, poria cocos polysaccharides, and total organic acids from hawthorn are collected in the water eluent; the ethanol gradient elution is as follows: 30% ethanol elution to collect the codonopsis glycoside fraction, 50% ethanol elution to collect the total glycoside fraction from Acanthopanax senticosus, 70% ethanol elution to collect the astragaloside A and atractylodes lactone III fractions, and the 90% ethanol eluent is discarded; the fractions are combined after their contents are detected by high performance liquid chromatography.

8. The use of the traditional Chinese medicine extract according to any one of claims 1 to 3 in the preparation of veterinary drugs or feed additives for relieving stunted growth in piglets.

9. The application according to claim 8, characterized in that: The term "stiffness" in piglets includes fetal stiffness, milk stiffness, food stiffness, and disease stiffness, especially growth retardation caused by intestinal villus atrophy and poor development of immune organs following diarrhea. When the term "Chinese herbal extract" is used as a feed additive, the amount added to the basic diet of piglets is 0.2% to 0.5% by weight, and the treatment course is 10 to 14 days.

10. A veterinary drug composition for relieving stunted growth in piglets, characterized in that: It contains an effective amount of the traditional Chinese medicine extract according to any one of claims 1 to 3, and a veterinary-acceptable carrier or excipient.

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