Active ingredients of Hu sheep and their application in immune-modulating health products

Abstract

This invention discloses an active ingredient from Huzhou sheep and its application in immunomodulatory health products, belonging to the field of immunomodulatory agent technology. Specifically, it relates to an active ingredient from Huzhou sheep, comprising Huzhou sheep placental extract and methyl galbaceous oxalate, with a mass ratio of 1:0.11-0.23. The Huzhou sheep active ingredient disclosed in this invention can effectively scavenge free radicals, reduce oxidative stress levels, and improve antioxidant capacity. Simultaneously, it enhances the positive regulation of phagocytic activity of phagocytes, thereby enhancing immune function and demonstrating good application value in immunomodulatory agents.

Description

Technical Field

[0001] This invention relates to the field of immunomodulatory technology, specifically to an active ingredient from Hu sheep and its application in immunomodulatory health products. Background Technology

[0002] Immune regulation is a core mechanism for maintaining health and defending against disease. Immunostimulants, also known as immunomodulators, are important drugs that can regulate, enhance, or restore the body's immune function. By acting on various aspects of the immune system, they play a crucial role in maintaining immune homeostasis and are widely used in the management of sub-health conditions, adjunctive treatment of infections, and clinical intervention for diseases related to immunodeficiency.

[0003] Traditional immunomodulatory drugs come from diverse sources, among which biologically derived preparations have attracted much attention due to their advantages such as mild effects and multi-target regulation. Placenta and its extracts, as an important source of bioactive substances, have shown potential in the field of immunomodulation. Placental peptides (or placental immunomodulatory factors, placental regulatory factors), as representative components, are characterized by small molecular weight, high bioactivity, and a broad immunomodulatory spectrum, capable of simultaneously promoting non-specific immunity, cellular immunity, and humoral immunity, demonstrating promising application prospects. However, the limited availability of human placenta and ethical concerns restrict its large-scale development and widespread application. Therefore, finding widely available, well-defined, and safe alternative sources of animal placenta has become one of the research directions in this field. Studies have shown that sheep placental peptides are highly similar to human placenta in nutritional composition and active ingredients, and are mainly composed of small-molecule polypeptides, rich in various amino acids and trace elements, exhibiting clear efficacy in enhancing immunity and tissue repair, and are considered ideal alternative raw materials.

[0004] Huzhou sheep not only possess excellent livestock economic value, but their placentas are also considered to have unique nutritional and medicinal potential due to their rich content of active ingredients, which are of positive significance for human health and disease adjuvant treatment. However, the systematic development and high-value utilization of active ingredients derived from Huzhou sheep, especially placental peptides, are still insufficient. Existing technologies mostly focus on the preparation of crude extracts from common sheep placentas, which suffers from problems such as unclear active ingredients, low standardization of preparation processes, and insufficient product efficacy stability, making it difficult to fully realize the potential advantages of Huzhou sheep as a unique resource in the preparation of highly effective and stable immunomodulators. Therefore, it is urgent to develop immunomodulators with clear components and stable activity based on the characteristics of Huzhou sheep placental extracts, so as to achieve high-value utilization of Huzhou sheep placental resources. Summary of the Invention

[0005] The purpose of this invention is to provide an active ingredient of Hu sheep and its application in immunomodulatory health products, which can effectively scavenge free radicals, reduce oxidative stress levels, improve antioxidant capacity, and enhance the positive regulation of phagocytic activity of phagocytes, thereby enhancing immune performance. It has good application value in immunomodulatory agents.

[0006] The technical solution adopted by the present invention to achieve the above objectives is as follows: An active ingredient from Hu sheep, characterized in that it comprises Hu sheep placental extract and methyl galacin; the mass ratio of the Hu sheep placental extract to methyl galacin is 1:0.11-0.23; the preparation method of the Hu sheep placental extract includes grinding and homogenizing the cotyledons of Hu sheep placenta, first ultrasonically treating it, then adding a compound enzyme and laurylamidopropyl dimethyl tertiary amine for enzymatic hydrolysis, centrifuging after enzymatic hydrolysis, taking the supernatant, rotary evaporating and concentrating it, and freeze-drying it to obtain the Hu sheep placental extract.

[0007] In the preparation of Hu sheep placenta extract, this invention combines neutral protease and papain, then adds laurylamidopropyl dimethyl tertiary amine to effectively improve enzymatic hydrolysis efficiency. This exposes amino acid residues and peptide chains with antioxidant capabilities, significantly enhancing the antioxidant capacity of the prepared Hu sheep placenta extract, reducing oxidative stress damage to phagocytes, and thus enhancing the phagocytic performance of phagocytes. Furthermore, the combined use of Hu sheep placenta extract and methyl arbutin promotes the absorption and targeted delivery of methyl arbutin. By neutralizing excess free radicals, it effectively enhances total antioxidant capacity, provides a stable internal environment for immune cells, protects the structure and function of immune cells, enhances the body's immune performance, significantly improves the phagocytic capacity of mononuclear phagocytes, and maintains the dynamic balance and long-term stability of the immune regulatory system.

[0008] Preferably, the complex enzyme includes neutral protease and papain.

[0009] Preferably, lauramidopropyl dimethyl tertiary amine is prepared by reacting lauric acid and dimethylaminopropylamine.

[0010] Preferably, the ultrasonic processing power is 200-600W.

[0011] Preferably, the ultrasonic treatment time is 10-30 minutes.

[0012] Preferably, the amount of enzyme added to the compound enzyme is 2000-6000 U / g.

[0013] Preferably, the enzymatic hydrolysis time is 1-4 hours.

[0014] Preferably, the enzymatic hydrolysis temperature is 30-50℃.

[0015] Preferably, the centrifugation rate is 2000-6000 r / min.

[0016] Preferably, the preparation method of lauramidopropyl dimethyl tertiary amine is as follows: Weigh out lauric acid, heat it to 60-80℃ and stir, add dimethylaminopropylamine, and react it under nitrogen protection at 140-160℃ for 6-12 hours. After the reaction is completed, cool it naturally to room temperature and purify it to obtain lauramidopropyl dimethyl tertiary amine.

[0017] More preferably, the molar ratio of lauric acid to dimethylaminopropylamine is 1:1-2.

[0018] Preferably, the preparation method of the placental extract of Hu sheep is as follows: Rinse the placenta of the Hu sheep with physiological saline, remove the cotyledons of the placenta and cut them into 0.5-2cm pieces. 3 Repeat the freeze-thaw cycle 2-5 times, grind and homogenize, first sonicate at 30-40℃ and 200-600W for 10-30 min, then add compound enzyme and lauramide propyl dimethyl tertiary amine and enzymatically hydrolyze at 30-50℃ for 1-4 h. After enzymatic hydrolysis, immediately place in a boiling water bath for 5-15 min to inactivate the enzyme, quickly cool to room temperature, centrifuge at 2000-6000 r / min for 20-40 min, collect the supernatant, concentrate by rotary evaporation, and freeze dry to obtain the Hu sheep placenta extract.

[0019] More preferably, the amount of enzyme added to the complex enzyme is 2000-6000 U / g.

[0020] More preferably, the complex enzyme includes neutral protease and papain.

[0021] More preferably, the enzyme ratio of papain to neutral protease is 1:1-4.

[0022] More preferably, the mass ratio of cotyledon to lauramide propyl dimethyl tertiary amine is 1:0.02-0.05.

[0023] Preferably, the active ingredients of the Hu sheep include Hu sheep placental extract and functional additives; the functional additives include at least one of gancissine A, lily of the valley saponin B and terpineol laurate.

[0024] More preferably, the active ingredients of the sheep include sheep placental extract and methyl guanylate.

[0025] More preferably, the mass ratio of the sheep placenta extract to methyl guanylate is 1:0.11-0.23.

[0026] More preferably, the active ingredients of the Hu sheep include Hu sheep placental extract, gancisic acid A, and lily of the valley saponin B. This invention combines Hu sheep placental extract, gancisic acid A, and lily of the valley saponin B, which may significantly enhance total antioxidant capacity by scavenging free radicals and activating antioxidant pathways, effectively protecting phagocytes from oxidative damage, thereby enhancing their phagocytic performance, strengthening immune function, and achieving sustained and stable immune regulation and immune system balance.

[0027] More preferably, the mass ratio of the sheep placenta extract to methyl guanylate is 1:0.11-0.23.

[0028] More preferably, the mass ratio of the sheep placenta extract to lily of the valley saponin B is 1:0.15-0.3.

[0029] More preferably, the active ingredients of the Hu sheep include Hu sheep placental extract, gancissine A, lily of the valley saponin B, and terpineol laurate. This invention further utilizes terpineol laurate in the active ingredients of the Hu sheep. Terpineol laurate is obtained by esterification modification of terpineol and lauric acid. The terpineol unit acts as an electron donor to neutralize reactive oxygen free radicals, while the lauric acid unit helps enhance the lipid solubility of the ingredient, making it easier to integrate into the cell membrane lipid environment, thereby further improving antioxidant capacity, reducing oxidative stress damage to phagocytes, and thus enhancing the phagocytic performance of phagocytes and the body's immune function.

[0030] More preferably, the mass ratio of the sheep placenta extract to methyl guanylate is 1:0.11-0.23.

[0031] More preferably, the mass ratio of the sheep placenta extract to lily of the valley saponin B is 1:0.15-0.3.

[0032] More preferably, the mass ratio of sheep placental extract to terpineol laurate is 1:0.015-0.05.

[0033] More preferably, the preparation method of terpineol laurate is as follows: Lauric acid and thionyl chloride were mixed and refluxed at 50-70℃ for 3-5 hours. The mixture was then distilled under reduced pressure, and triethylamine and terpineol were added. The mixture was then reacted at 60-80℃ for 2-6 hours. After the reaction was completed, the mixture was extracted with ethyl acetate, and the pH was adjusted to neutral with sodium bicarbonate solution. The organic layer was separated, dried, filtered, and rotary evaporated. After purification by column chromatography, terpineol laurate was obtained.

[0034] More preferably, the molar ratio of lauric acid to thionyl chloride is 1:0.5-1.5.

[0035] More preferably, the ratio of lauric acid to triethylamine is 1 mol: 50-200 mL.

[0036] More preferably, the molar ratio of lauric acid to terpineol is 1:0.2-1.

[0037] More preferably, the temperature of the sodium bicarbonate solution is 0-10°C.

[0038] This invention also discloses the application of active ingredients from Hu sheep in the preparation of immunomodulatory agents.

[0039] This invention, by combining Hu sheep placental extract with Ganxi sage acid A, lily of the valley saponin B, and terpineol laurate to obtain Hu sheep active ingredients, has the following beneficial effects: The Hu sheep active ingredients disclosed in this invention can not only scavenge free radicals and improve total antioxidant capacity, exhibiting excellent antioxidant properties; they can also significantly enhance the phagocytic function of phagocytes, increase the phagocytic index of phagocytes, and enhance the body's immune function. Therefore, this invention discloses a Hu sheep active ingredient with good antioxidant and immune-enhancing properties, showing significant application prospects in the development and application of immunomodulators. Attached Figure Description

[0040] Figure 1 This refers to enzyme activity.

[0041] Figure 2 Electrophoresis diagram of the placental extract from Hu sheep.

[0042] Figure 3 This is a chromatography separation diagram.

[0043] Figure 4 The value represents the DPPH free radical scavenging rate.

[0044] Figure 5 This is the consuming index. Detailed Implementation

[0045] 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.

[0046] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0047] Example 1: The preparation method of lauramide propyl dimethyl tertiary amine includes, Lauric acid was weighed, heated to 70°C and stirred, and dimethylaminopropylamine was added. The mixture was reacted at 150°C for 8 hours under nitrogen protection. After the reaction was completed, it was naturally cooled to room temperature and purified to obtain lauramidopropyl dimethyl tertiary amine. The molar ratio of lauric acid to dimethylaminopropylamine was 1:1.

[0048] The preparation method of Hu sheep placenta extract includes, Rinse the placenta of the Hu sheep with physiological saline, remove the cotyledons of the placenta and cut them into 1cm pieces. 3 The mixture was repeatedly freeze-thawed three times, ground into a homogenate, and then sonicated at 35℃ and 420W for 15 min. Then, a compound enzyme and lauramide propyl dimethyl tertiary amine were added, and the mixture was enzymatically hydrolyzed at 40℃ for 2 h. After hydrolysis, the mixture was immediately placed in a boiling water bath for 10 min to inactivate the enzyme, rapidly cooled to room temperature, and centrifuged at 4000 r / min for 30 min. The supernatant was collected, concentrated by rotary evaporation, and freeze-dried to obtain the extract of sheep placenta. The compound enzyme dosage was 4000 U / g, and the compound enzyme consisted of neutral protease and papain, with a papain to neutral protease enzyme ratio of 1:2; the mass ratio of the compound enzyme to lauramide propyl dimethyl tertiary amine was 1:0.05.

[0049] The active ingredients of the sheep include sheep placenta extract and methyl galbana. The mass ratio of sheep placenta extract to methyl galbana is 1:0.23.

[0050] Example 2: The preparation method of lauramide propyl dimethyl tertiary amine is the same as in Example 1.

[0051] The preparation method of the placental extract of sheep from Huzhou is the same as that in Example 1, except that the mass ratio of the compound enzyme and lauramide propyl dimethyl tertiary amine is changed to 1:0.02.

[0052] The active ingredients of the sheep include sheep placenta extract and methyl galbana. The mass ratio of sheep placenta extract to methyl galbana is 1:0.23.

[0053] Example 3: The preparation method of lauramide propyl dimethyl tertiary amine is the same as in Example 1.

[0054] The preparation method of the placental extract from Hu sheep is the same as in Example 1.

[0055] The active ingredients of the sheep include sheep placenta extract and methyl galbana. The mass ratio of sheep placenta extract to methyl galbana is 1:0.11.

[0056] Example 4: The preparation method of lauramide propyl dimethyl tertiary amine is the same as in Example 1.

[0057] The preparation method of the placental extract from Hu sheep is the same as in Example 1.

[0058] The active ingredients of the sheep include sheep placenta extract and methyl galbana. The mass ratio of sheep placenta extract to methyl galbana is 1:0.05.

[0059] Example 5: The preparation method of lauramide propyl dimethyl tertiary amine is the same as in Example 1.

[0060] The preparation method of the placental extract from Hu sheep is the same as in Example 1.

[0061] The active ingredients of the sheep include sheep placenta extract and methyl galbana. The mass ratio of sheep placenta extract to methyl galbana is 1:0.4.

[0062] Example 6: The preparation method of lauramide propyl dimethyl tertiary amine is the same as in Example 1.

[0063] The preparation method of the placental extract from Hu sheep is the same as in Example 1.

[0064] The active ingredients of the sheep include sheep placenta extract, gancisic acid A, and lily of the valley saponin B. The mass ratio of sheep placenta extract to gancisic acid A is 1:0.23; the mass ratio of sheep placenta extract to lily of the valley saponin B is 1:0.05.

[0065] Example 7: The preparation method of lauramide propyl dimethyl tertiary amine is the same as in Example 1.

[0066] The preparation method of the placental extract from Hu sheep is the same as in Example 1.

[0067] The active ingredients of the sheep include sheep placenta extract, gancisic acid A, and lily of the valley saponin B. The mass ratio of sheep placenta extract to gancisic acid A is 1:0.23; the mass ratio of sheep placenta extract to lily of the valley saponin B is 1:0.02.

[0068] Example 8: The preparation method of lauramide propyl dimethyl tertiary amine is the same as in Example 1.

[0069] The preparation method of the placental extract from Hu sheep is the same as in Example 1.

[0070] Methods for preparing terpineol laurate include, Lauric acid and thionyl chloride were mixed and refluxed at 60°C for 4 hours. The mixture was then distilled under reduced pressure, followed by the addition of triethylamine and terpineol. The reaction was carried out at 70°C for 4 hours. After the reaction was complete, the mixture was extracted with ethyl acetate, and the pH was adjusted to neutral with saturated sodium bicarbonate solution. The organic layer was separated, dried, filtered, and rotary evaporated. The resulting product was purified by column chromatography to obtain terpineol laurate. The molar ratio of lauric acid to thionyl chloride was 1:0.75; the molar ratio of lauric acid to triethylamine was 1 mol:100 mL; the molar ratio of lauric acid to terpineol was 1:0.5; and the temperature of the saturated sodium bicarbonate solution was 4°C.

[0071] The active ingredients of the Hu sheep include Hu sheep placenta extract, gancissine A, lily of the valley saponin B, and terpineol laurate. The mass ratio of Hu sheep placenta extract to gancissine A is 1:0.23; the mass ratio of Hu sheep placenta extract to lily of the valley saponin B is 1:0.05; and the mass ratio of Hu sheep placenta extract to terpineol laurate is 1:0.3.

[0072] Example 9: The preparation method of lauramide propyl dimethyl tertiary amine is the same as in Example 1.

[0073] The preparation method of the placental extract from Hu sheep is the same as in Example 1.

[0074] The preparation method of terpineol laurate is the same as in Example 8.

[0075] The active ingredients of the sheep include sheep placenta extract, gancissine A, lily of the valley saponin B, and terpineol laurate. The mass ratio of sheep placenta extract to gancissine A is 1:0.23; the mass ratio of sheep placenta extract to lily of the valley saponin B is 1:0.05; and the mass ratio of sheep placenta extract to terpineol laurate is 1:0.15.

[0076] Comparative Example 1: The preparation method of the placental extract of sheep from Huzhou is the same as that in Example 1, except that lauroamide propyl dimethyl tertiary amine was not used.

[0077] Comparative Example 2: The preparation method of the placental extract of sheep from Huzhou is the same as that in Example 1, except that lauroamide propyl dimethyl tertiary amine is replaced with lauric acid.

[0078] Comparative Example 3: The preparation method of the placental extract from Hu sheep is the same as in Example 1.

[0079] The active ingredients of Hu sheep include Hu sheep placental extract.

[0080] Comparative Example 4: The preparation method of the placental extract from Hu sheep is the same as in Example 1.

[0081] The active ingredients of Hu sheep include methyl gancisic acid.

[0082] Comparative Example 5: The preparation method of the placental extract from Hu sheep is the same as in Example 1.

[0083] The active ingredients of Hu sheep include lily of the valley saponin B.

[0084] Comparative Example 6: The preparation method of the placental extract from Hu sheep is the same as in Example 1.

[0085] The active ingredients of Hu sheep include terpineol laurate.

[0086] Comparative Example 7: The preparation method of the placental extract from Hu sheep is the same as in Example 1.

[0087] The active ingredients of the sheep include sheep placenta extract and lily of the valley saponin B. The mass ratio of sheep placenta extract to lily of the valley saponin B is 1:0.05.

[0088] Comparative Example 8: The preparation method of the placental extract from Hu sheep is the same as in Example 1.

[0089] The preparation method of terpineol laurate is the same as in Example 8.

[0090] The active ingredients of the sheep include sheep placenta extract and terpineol laurate. The mass ratio of sheep placenta extract to terpineol laurate is 1:0.3.

[0091] Experimental example: 1. Enzyme activity Experimental groups were set up: The complex enzyme was divided into 7 groups and mixed with laurylamidopropyl dimethyl tertiary amine at mass ratios of 1:0, 1:0.03, 1:0.05, 1:0.1, 1:0.15, 1:0.2 and 1:0.25, respectively. The complex enzyme included neutral protease and papain, with the ratio of papain to neutral protease being 1:2. Phosphate buffer was added to 1.5 mL for each group and treated at 37℃ for 10 min. Subsequently, 500 μL of 10 g / L casein solution was added to each group and reacted at 37℃ for 10 min. The activity of the complex enzyme was determined using a protease activity assay kit purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0092] A negative control group was set up: no compound enzyme and lauramide propyl dimethyl tertiary amine were added, 1.5 mL of phosphate buffer and 500 μL of casein solution with a concentration of 10 g / L were added, and the reaction was carried out at 37 °C for 10 min. The activity of the compound enzyme was measured using a protease activity assay kit purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0093] A positive control group was set up: The complex enzyme, consisting of neutral protease and papain, was treated at 37°C for 10 min. The ratio of papain to neutral protease was 1:2. Phosphate buffer was added to the complex enzyme to a final volume of 1.5 mL, and the mixture was treated at 37°C for 10 min. Subsequently, 500 μL of 10 g / L casein solution was added to each group, and the reaction was carried out at 37°C for 10 min. The activity of the complex enzyme was measured using a protease activity assay kit purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0094] The enzyme activity in the negative control group was 0%, indicating the reliability of the experimental system. Under the condition of no addition of compound enzyme, the other reagents used in the experimental system did not produce catalytic reactions, thus eliminating the interference of non-enzymatic reactions or reagents themselves on the experimental results.

[0095] The enzyme activity of the complex enzyme without the addition of laurylamide propyl dimethyl tertiary amine was defined as 100%, that is, the enzyme activity of the positive control group was defined as 100%. The enzyme activity results of the experimental group are as follows: Figure 1 As shown, with the increase of the amount of lauramidopropyl dimethyl tertiary amine used, the enzyme activity exhibited a trend of first increasing and then decreasing. The enzyme activity was highest when the mass ratio of the compound enzyme to lauramidopropyl dimethyl tertiary amine was 1:0.05. Within a lower concentration range, lauramidopropyl dimethyl tertiary amine may enhance the binding to the substrate and catalytic efficiency by fine-tuning the conformation of the enzyme protein's active site, thereby promoting the enzymatic reaction; however, when the concentration exceeds a certain range, it may inhibit the enzymatic reaction by disrupting the protein structure. Therefore, a mass ratio of 1:0.05 for the compound enzyme and lauramidopropyl dimethyl tertiary amine can be selected for subsequent experiments.

[0096] The complex enzyme was divided into two groups, designated as treatment group 1 and treatment group 2. The complex enzyme consisted of neutral protease and papain, with a papain to neutral protease ratio of 1:2. In treatment group 1, the complex enzyme was mixed with laurylamidopropyl dimethyl tertiary amine at a mass ratio of 1:0.05; in treatment group 2, the complex enzyme was mixed with lauric acid at a mass ratio of 1:0.05. Phosphate buffer was then added to each group to a final volume of 1.5 mL, and the mixture was incubated at 37°C for 10 min. Subsequently, 500 μL of a 10 g / L casein solution was added to each group, and the mixture was incubated at 37°C for 10 min. The activity of the complex enzyme was determined using a protease activity assay kit purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0097] The enzyme activity of the positive control group was defined as 100%. The enzyme activity of treatment group 1 was 156%, and the enzyme activity of treatment group 2 was basically the same as that of the positive control group. This indicates that lauramidopropyl dimethyl tertiary amine is more effective in promoting enzymatic reactions than lauric acid.

[0098] 2. Degree of enzymatic hydrolysis Referring to the preparation method of the placental extract of Hu sheep in Example 1, the placenta of Hu sheep was rinsed clean with physiological saline, and the cotyledons of the placenta were taken and cut into 1cm pieces. 3 The mixture was repeatedly frozen and thawed three times, then ground into a homogenate. The total nitrogen content was determined using the micro-Kjeldahl method and recorded as TN. The mixture was first sonicated at 35℃ and 420W for 15 min, then a compound enzyme and lauramide propyl dimethyl tertiary amine were added, and the mixture was enzymatically hydrolyzed at 40℃ for 3 h. After the enzymatic hydrolysis was completed, the mixture was immediately placed in a boiling water bath for 10 min to inactivate the enzyme, then rapidly cooled to room temperature, and centrifuged at 4000 r / min for 30 min. The supernatant was collected, and the ammonia nitrogen content was determined using the formaldehyde titration method and recorded as SN1.

[0099] The total nitrogen content and ammonia nitrogen content of the sheep placenta extracts in Example 2, Comparative Example 1, and Comparative Example 2 were determined according to the above-described methods for determining total nitrogen content and ammonia nitrogen content.

[0100] A control group was set up. The placenta of Hu sheep was rinsed with physiological saline, and the cotyledons of the placenta were taken and cut into 1cm pieces. 3 The mixture was repeatedly frozen and thawed three times, then ground into a homogenate. It was first sonicated at 35℃ and 420W for 15 minutes, then enzymatically hydrolyzed at 40℃ for 2 hours. After the enzymatic hydrolysis, it was immediately placed in a boiling water bath for 10 minutes to inactivate the enzyme, then rapidly cooled to room temperature, and centrifuged at 4000 r / min for 30 minutes. The supernatant was collected, and the ammonia nitrogen content was determined by formaldehyde titration, denoted as SN0.

[0101] The degree of hydrolysis is calculated using the following formula: Degree of hydrolysis (%) = (SN1 - SN0) / TN × 100%.

[0102] Table 1 Degree of hydrolysis

[0103] The results are shown in Table 1. The degree of hydrolysis in Examples 1-2 was significantly higher than that in Comparative Examples 1 and 2. This is because, in the preparation method of the sheep placenta extract, Example 1 used lauramidopropyl dimethyl tertiary amine to assist enzymatic hydrolysis, while Comparative Example 1 did not use lauramidopropyl dimethyl tertiary amine, and Comparative Example 2 used lauric acid. The results indicate that the use of lauramidopropyl dimethyl tertiary amine can effectively promote the enzymatic reaction, thereby significantly increasing the degree of hydrolysis during the enzymatic hydrolysis process, which helps to produce small molecule peptides and exposes amino acid residues and peptide chains with antioxidant capabilities.

[0104] 3. Electrophoresis The placental extract of the sheep prepared in Example 1 was preliminarily identified by SDS-PAGE gel electrophoresis.

[0105] Figure 2 This is an electrophoresis image of the extract from the placenta of Hu sheep. The results are as follows: Figure 2 As shown in the SDS-PAGE gel electrophoresis image, four distinct peptide bands are mainly displayed, with molecular weights of 10 kDa, 8 kDa, 7.5 kDa, and 6.5 kDa from top to bottom. This indicates that the placental peptides extracted from the placenta of the Hu sheep are a mixture of polypeptides.

[0106] 4. Identification by gel chromatography The placental extract of sheep prepared in Example 1 was subjected to gel chromatography using Superdex-75 at a flow rate of 1 mL / min and a sample loading volume of 1 mL. The UV absorption wavelength was 220 nm. After pressure line equilibration, the sample was loaded into the sample cell and collected according to the UV absorption peak.

[0107] Figure 3 This is a chromatography separation diagram. The results are as follows: Figure 3 As shown, gel chromatography separation of the sheep placenta extract yielded four separate fractions, a result largely consistent with electrophoresis. This indicates that the sheep placental peptides are a mixture containing four components.

[0108] 5. Amino acid composition analysis The amino acid composition and content in the placental extract of sheep prepared in Example 1 were determined by high performance liquid chromatography, and the mass fraction of each amino acid in the total amino acids was calculated.

[0109] Table 2. Amino acid composition and its mass fraction

[0110] Seventeen amino acids were detected in the extract of Hu sheep placenta, of which essential amino acids accounted for 36.67% of the total amino acids. This indicates that the extract of Hu sheep placenta has good nutritional value.

[0111] 6. Total antioxidant capacity determination The active ingredients of the Hu sheep from Examples 1-9 and Comparative Examples 1-8 were mixed with dimethyl sulfoxide (DMSO), wherein the ratio of Hu sheep placental extract to DMSO in the active ingredients was 1 mg:0.25 mL, to obtain the Hu sheep active ingredient solutions of Examples 1-9 and Comparative Examples 1-8. The antioxidant capacity of the Hu sheep active ingredient solutions of Examples 1-9 and Comparative Examples 1-8 was determined using the DPPH method, following the instructions of the DPPH free radical scavenging ability test kit. The DPPH free radical scavenging ability test kit was purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0112] Figure 4 The DPPH free radical scavenging rate is shown in the results. Figure 4 As shown, compared with Comparative Examples 1-2, the DPPH radical scavenging rate of Examples 1-2 was significantly improved. This is because in the preparation method of the Hu sheep placenta extract, Examples 1-2 used lauramidopropyl dimethyl tertiary amine to assist enzymatic hydrolysis, while Comparative Example 1 did not use lauramidopropyl dimethyl tertiary amine, and Comparative Example 2 used lauric acid. The results indicate that using an appropriate amount of lauramidopropyl dimethyl tertiary amine can effectively improve the enzymatic hydrolysis efficiency, exposing amino acid residues and peptide chains with antioxidant capabilities, thereby significantly enhancing the antioxidant capacity of the prepared Hu sheep placenta extract.

[0113] Compared to Comparative Examples 3-6, the DPPH free radical scavenging rates of Examples 1-9 of this invention were significantly improved. This is because, in the active ingredients of the sheep, Comparative Example 3 used only sheep placental extract, Comparative Example 4 used only methyl galbapple, Comparative Example 5 used only lily of the valley saponin B, and Comparative Example 6 used only terpineol laurate. In contrast, Examples 1-9, in addition to using sheep placental extract, also added at least one of methyl galbapple, lily of the valley saponin B, and terpineol laurate. This indicates that compared to using sheep placental extract, methyl galbapple, lily of the valley saponin B, or terpineol laurate alone, combining sheep placental extract with at least one of methyl galbapple, lily of the valley saponin B, and terpineol laurate results in a more effective antioxidant capacity of the sheep active ingredients.

[0114] Compared with Example 1 and Comparative Examples 7-8, the DPPH free radical scavenging rate of Examples 6-9 was further improved. This is because, in the active ingredients of the sheep, Example 1 used a combination of sheep placental extract and galvanic acid, Comparative Example 7 used a combination of sheep placental extract and lily of the valley saponin B, and Comparative Example 8 used a combination of sheep placental extract and terpineol laurate; while Examples 6-7 used a combination of sheep placental extract with galvanic acid and lily of the valley saponin B, and Examples 8-9 used a combination of sheep placental extract with galvanic acid, lily of the valley saponin B, and terpineol laurate. This indicates that compared to using Ganci salsaponin A, lily of the valley saponin B, or terpineol laurate alone in combination with Hu sheep placental extract, the antioxidant capacity of the Hu sheep active ingredients obtained by using at least two of the following components is better: Ganci salsaponin A, lily of the valley saponin B, and terpineol laurate. Furthermore, the antioxidant capacity of the Hu sheep active ingredients obtained by using all three components in combination is the best.

[0115] Based on the experimental data from Examples 1-9, it is evident that the antioxidant capacity of the active ingredient from Hu sheep obtained by combining appropriate amounts of senna methyl arbutin, lily of the valley saponin B, and terpineol laurate with Hu sheep placental extract is better.

[0116] 7. Carbon Purification Experiment The effects of the active ingredients of Hu sheep in Examples 1-9 and Comparative Examples 1-8 on the phagocytic function of mononuclear macrophages were determined using the carbon clearance method. The specific steps are as follows: S1. Mice were randomly divided into a blank control group, a positive control group, a solvent control group, and 17 experimental groups, with 3 mice in each group.

[0117] S2. The active ingredients of Hu sheep in Examples 1-9 and Comparative Examples 1-8 were mixed with dimethyl sulfoxide to prepare Hu sheep active ingredient solutions with a concentration of 2 mg / mL for Examples 1-9 and Comparative Examples 1-8, respectively.

[0118] S3. In the blank control group, 0.2 mL / animal of physiological saline was administered by gavage; in the positive control group, 0.2 mL / animal of porcine spleen transfer factor was administered by gavage; in the solvent control group, 0.2 mL / animal of dimethyl sulfoxide was administered by gavage; and in the 17 experimental groups, 0.2 mL / animal of the active ingredient solution of Hu sheep from Examples 1-9 and Comparative Examples 1-8 was administered by gavage, respectively.

[0119] S4. Each group was administered the drug continuously for 5 days, once daily by gavage. One hour after each gavage administration, Indian ink was injected into the tail vein of the mice. The mass concentration of Indian ink was 20%, and the dosage was 0.01 mL / kg. At 1 minute and 5 minutes after injection, 20 μL of blood was collected from the orbital cavity of the mice. 2.5 mL of 10% sodium carbonate solution was added, and the mixture was shaken well. The absorbance was measured at 600 nm, and the phagocytic index was calculated. The formula for calculating the phagocytic index is: Phagocytic index = (logOD1 - logOD2) / (t2 - t1), where OD1 is the absorbance value of the blood sample at 1 minute, OD2 is the absorbance value of the blood sample at 5 minutes, and t1 is 1 minute and t2 is 5 minutes.

[0120] Porcine spleen transfer factor is an immunomodulatory biological agent known to nonspecifically activate the mononuclear macrophage system. It significantly enhances the phagocytic index in carbon clearance assays and is therefore often used as a positive control. Porcine spleen transfer factor has a well-defined immunomodulatory effect, providing a reliable reference for experimental systems. It can be used to assess the enhancing effect of analytes on phagocytic function of phagocytes and to exclude false negative results.

[0121] Figure 5 The phagocytic index. The results are as follows: Figure 5 As shown, compared with the blank control group, the phagocytic index of the positive control group was significantly increased, indicating that the experimental system of the present invention is effective and sensitive, and can detect the immune enhancement effect. Compared with the blank control group, the positive control group, and the solvent control group, the phagocytic index of Examples 1-9 and Comparative Examples 1-8 of the present invention was significantly increased, indicating that the active ingredient of the sheep in the present invention can increase the phagocytic index of mononuclear phagocytes and has a good immune effect.

[0122] Compared with Comparative Examples 1-2, the phagocytic index of Examples 1-2 was significantly improved. This is because in the preparation method of the Hu sheep placenta extract, Examples 1-2 used lauramidopropyl dimethyl tertiary amine to assist enzymatic hydrolysis, while Comparative Example 1 did not use lauramidopropyl dimethyl tertiary amine, and Comparative Example 2 used lauric acid. The results indicate that using an appropriate amount of lauramidopropyl dimethyl tertiary amine can effectively improve the enzymatic hydrolysis efficiency, exposing amino acid residues and peptide chains with antioxidant capabilities, significantly enhancing the antioxidant capacity of the prepared Hu sheep placenta extract, reducing oxidative stress damage to phagocytes, and thus enhancing the phagocytic performance of phagocytes and the body's immune function. Compared to Comparative Examples 3-6, the phagocytic index of Examples 1-9 of this invention was significantly improved. This is because, in the active ingredients of the Hu sheep, Comparative Example 3 used only Hu sheep placental extract, Comparative Example 4 used only Gancieryl methyl arbutin, Comparative Example 5 used only lily of the valley saponin B, and Comparative Example 6 used only terpineol laurate. In contrast, Examples 1-9, in addition to using Hu sheep placental extract, also added at least one of Gancieryl methyl arbutin, lily of the valley saponin B, and terpineol laurate. This indicates that, compared to using Hu sheep placental extract alone, combining Hu sheep placental extract with at least one of Gancieryl methyl arbutin, lily of the valley saponin B, and terpineol laurate resulted in a better immune effect of the Hu sheep active ingredients.

[0123] Compared with Example 1 and Comparative Examples 7-8, the phagocytosis index of Examples 6-9 was further improved because, in the active ingredients of the sheep, Example 1 used a combination of sheep placental extract and galvanic acid, Comparative Example 7 used a combination of sheep placental extract and lily of the valley saponin B, and Comparative Example 8 used a combination of sheep placental extract and terpineol laurate; while Examples 6-7 used a combination of sheep placental extract with galvanic acid and lily of the valley saponin B, and Examples 8-9 used a combination of sheep placental extract with galvanic acid, lily of the valley saponin B, and terpineol laurate. This indicates that compared to using Ganxi sage, lily of the valley saponin B, or terpineol laurate alone in combination with Hu sheep placental extract, the immune efficacy of the Hu sheep active ingredient is better when using at least two of the following components: Ganxi sage, lily of the valley saponin B, and terpineol laurate. Furthermore, the immune efficacy of the Hu sheep active ingredient is best when all three components are used in combination.

[0124] Based on the experimental data from Examples 1-9, it is evident that the immune-boosting effect of the active ingredients from Hu sheep obtained by combining appropriate amounts of gancissine A, lily of the valley saponin B, and terpineol laurate with Hu sheep placental extract is better.

[0125] The conventional operations in the operation steps of this invention are well known to those skilled in the art and will not be described in detail here.

[0126] The embodiments described above provide a detailed explanation of the technical solutions of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any changes and modifications made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An active ingredient from Hu sheep, characterized in that, The product includes a placental extract of Huzhou sheep and methyl galacin; the mass ratio of the placental extract of Huzhou sheep to methyl galacin is 1:0.11-0.23; the preparation method of the placental extract of Huzhou sheep includes grinding and homogenizing the cotyledons of the placenta of Huzhou sheep, first sonicating, then adding a compound enzyme and laurylamidopropyl dimethyl tertiary amine for enzymatic hydrolysis, after enzymatic hydrolysis, centrifuging, taking the supernatant, rotary evaporating and concentrating, and freeze-drying to obtain the placental extract of Huzhou sheep.

2. The active ingredient of Hu sheep according to claim 1, characterized in that, The complex enzyme includes neutral protease and papain.

3. The active ingredient of Hu sheep according to claim 1, characterized in that, The lauramidopropyl dimethyl tertiary amine is prepared by reacting lauric acid and dimethylaminopropylamine.

4. The active ingredient of Hu sheep according to claim 1, characterized in that, The ultrasonic processing power is 200-600W.

5. The active ingredient of Hu sheep according to claim 1, characterized in that, The ultrasonic treatment time is 10-30 minutes.

6. The active ingredient of Hu sheep according to claim 1, characterized in that, The amount of the compound enzyme added is 2000-6000 U / g.

7. The active ingredient of Hu sheep according to claim 1, characterized in that, The enzymatic hydrolysis time is 1-4 hours.

8. The active ingredient of Hu sheep according to claim 1, characterized in that, The enzymatic hydrolysis temperature is 30-50℃.

9. The active ingredient of Hu sheep according to claim 1, characterized in that, The centrifugation rate is 2000-6000 r / min.

10. The use of the active ingredient of the lake sheep as described in claim 1 in the preparation of immunomodulatory agents.

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