Herbal composition containing pancreatic peptide and bone marrow peptide and preparation and application thereof

Abstract

The application relates to the field of biological medicine, and discloses a herbal composition containing pancreatic peptide and bone marrow peptide as well as preparation and application of the herbal composition. The composition comprises pancreatic peptide, bone marrow peptide and herbal extract. The pancreatic peptide is obtained by low-temperature pretreatment, endogenous protease action and further hydrolysis of animal pancreatic tissue; the bone marrow peptide is obtained by protein extraction, enzymolysis and separation and purification of animal bone marrow tissue; and the herbal extract is obtained by extraction treatment of herbal raw materials. The pancreatic peptide obtained through a specific degradation process is compounded with the bone marrow peptide and the herbal extract, different source active components form a composite system, the application effect of the animal source peptide in the composition is improved, the obtained composition has good application performance, and can be used for preparing related products for inhibiting proliferation of pancreatic cancer cells.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a herbal composition containing pancreatic peptides and bone marrow peptides, and its preparation and application. Background Technology

[0002] Animal-derived peptides are a class of peptides obtained from animal tissues, organs, or related protein resources through extraction, hydrolysis, and other processing methods. Due to the abundance of animal tissues, peptides with different molecular weight distributions and amino acid composition characteristics can be formed after protein degradation. In recent years, they have been increasingly used in nutritional supplements and functional foods. During the preparation of animal-derived peptides, the protein hydrolysis method affects the location of protein chain breakage, peptide length distribution, and the final composition of the peptide components. Therefore, obtaining animal-derived peptides with specific compositional characteristics is of great significance for improving the performance of animal-derived peptide products.

[0003] In the prior art, relevant research has been carried out on the preparation of animal-derived peptides and the compound application of animal-derived peptides with plant active ingredients. For example, Chinese patent CN108208496A discloses a method for preparing a hypoglycemic polypeptide herbal solid beverage and liver peptide powder. This technology combines bone marrow peptide powder, liver peptide powder and various herbal extracts to form a composite product containing animal-derived peptides and plant-derived ingredients. Chinese patent CN101716188B discloses an animal pancreas extract for the prevention and treatment of diabetes. This technology uses animal pancreas tissue as raw material and obtains small molecule active substances from animal pancreas through extraction and processing. Chinese patent CN109371089A discloses a method for extracting small molecule liver peptides. This technology obtains small molecule peptide products through animal tissue disruption, protease hydrolysis and separation and purification, providing a technical solution for the preparation of animal-derived peptides.

[0004] However, in existing animal-derived peptide complex products, animal tissue proteins are usually directly hydrolyzed by exogenous enzymes to form peptide components. The protein degradation process is mainly determined by the characteristics of the exogenous enzymes themselves. The molecular composition characteristics and component states of the peptide components are limited by a single enzymatic hydrolysis process. When animal-derived peptides are further used as components in a complex functional system, the existing technology usually combines them with other functional components after obtaining the animal-derived peptides. The lack of a design link between the formation process of animal-derived peptides and the complex system makes it difficult to obtain peptide component states that match the complex system when animal-derived peptides are used as components of a complex functional system. Summary of the Invention

[0005] The technical problem to be solved by this invention is: how to improve the limitation of peptide component state by a single enzymatic hydrolysis process in the formation of animal-derived peptides, so as to match the composition state of animal-derived peptides with the component requirements of complex functional systems. To this end, this invention provides a herbal composition containing pancreatic peptides and bone marrow peptides, as well as its preparation and application.

[0006] To achieve the above objectives, the present invention provides an herbal composition containing pancreatic peptides and bone marrow peptides, comprising pancreatic peptides, bone marrow peptides, and herbal extracts.

[0007] The pancreatic peptides are prepared from animal pancreatic tissue through low-temperature pretreatment, self-activation, ethanol-assisted activation, autolysis, and further hydrolysis by exogenous proteases.

[0008] In the preparation of the pancreatic peptides, the animal pancreatic tissue is first subjected to low-temperature treatment, which allows endogenous proteases in the pancreatic tissue to gradually participate in the protein degradation process. Then, through self-activation, ethanol-assisted activation, and autolysis, the endogenous proteases act on the pancreatic tissue proteins to form a pre-degradation system. Subsequently, exogenous proteases are used to further hydrolyze the pre-degradation system to obtain pancreatic peptide components that are different from those obtained by direct hydrolysis with exogenous proteases.

[0009] The bone marrow peptides were obtained from animal bone marrow tissue through protein extraction, enzymatic hydrolysis, and separation and purification.

[0010] Furthermore, the preparation process of the pancreatic peptide includes: subjecting animal pancreatic tissue to low-temperature disruption to obtain pancreatic tissue slurry; subjecting the pancreatic tissue slurry to self-activation treatment, so that endogenous proteases gradually participate in the protein degradation process; subsequently, through activation and autolysis treatment, allowing the endogenous proteases to act on the protein substrate in the pancreatic tissue to form a protein degradation system pretreated with endogenous proteases; and then adding exogenous proteases to the protein degradation system to further hydrolyze the protein substrate treated with endogenous proteases to obtain pancreatic peptide.

[0011] The endogenous protease is an endogenous protease system naturally present in animal pancreatic tissue, and is not a single protease preparation added separately during the preparation process. The endogenous protease system includes trypsin, chymotrypsin, elastase, and carboxypeptidase. The above proteases mainly exist in the form of their respective zymogens in animal pancreatic tissue.

[0012] The endogenous protease system participates in the degradation of pancreatic tissue proteins after low-temperature lysis of animal pancreatic tissue through self-activation, ethanol-assisted activation, and autolysis, forming a protein degradation system for further hydrolysis by papain.

[0013] Furthermore, in the preparation of the pancreatic peptide, the endogenous protease action stage and the exogenous protease hydrolysis stage are continuously connected. The exogenous protease does not act directly on the untreated animal tissue protein, but acts on the protein degradation system after self-activation and autolysis, thereby changing the degradation process of animal tissue protein.

[0014] Furthermore, the bone marrow peptide is obtained from animal bone marrow tissue through protein extraction, enzymatic hydrolysis, and separation and purification. The bone marrow peptide is used as an animal-derived active peptide component to construct a complex functional system together with pancreatic peptides.

[0015] Furthermore, the herbal extract is composed of ginseng, astragalus, dandelion, polygonatum, and saffron, and is used to form a complex composition containing animal-derived active peptides and plant-derived active ingredients together with pancreatic peptides and bone marrow peptides.

[0016] The present invention also provides a method for preparing the above-mentioned herbal composition containing pancreatic peptides and bone marrow peptides, comprising the following steps: S1: Animal pancreatic tissue is subjected to low-temperature disruption, self-activation, ethanol-assisted activation, and autolysis to enable endogenous proteases in the pancreatic tissue to participate in the protein degradation process. Subsequently, exogenous proteases are added for further hydrolysis to obtain pancreatic peptides. S2: Bone marrow peptides are obtained from animal bone marrow tissue through protein extraction, enzymatic hydrolysis, and separation and purification. S3: Extracting herbal raw materials to obtain herbal extracts; S4: Mix the obtained pancreatic peptides, bone marrow peptides and herbal extracts to form a complex functional system with the pancreatic peptides obtained through a special degradation process and the bone marrow peptides and herbal extracts, thereby obtaining a herbal composition containing pancreatic peptides and bone marrow peptides.

[0017] The present invention also provides the use of the above-mentioned herbal composition containing pancreatic peptides and bone marrow peptides in the preparation of products for inhibiting the proliferation of pancreatic cancer cells.

[0018] The technical effects and advantages of this invention are as follows: In this invention, by subjecting animal pancreatic tissue to low-temperature pretreatment, self-activation, ethanol-assisted activation, and autolysis, endogenous proteases in the pancreatic tissue are pre-involved in the protein degradation process. Further hydrolysis is then performed using exogenous proteases. This method differs from existing techniques that directly hydrolyze animal tissue proteins using exogenous proteases. The resulting pancreatic peptides possess compositional characteristics and activity levels distinct from conventional animal-derived peptides. Furthermore, the pancreatic peptides are combined with bone marrow peptides and herbal extracts to form a synergistic composition system with multiple active components. This combination of endogenous protease pretreatment and deep hydrolysis with exogenous proteases enhances the utilization of animal tissue proteins, resulting in a composition that exhibits superior efficacy in inhibiting pancreatic cancer cell proliferation. Simultaneously, it addresses the limitations of existing animal-derived peptides, which often have a single source of active components and limited efficacy. This invention obtains pancreatic peptides with specific active characteristics through a specific preparation process and combines them with bone marrow peptides and herbal extracts, thereby improving the overall application performance of the composition. Attached Figure Description

[0019] The disclosure of this invention is illustrated with reference to the accompanying drawings. It should be understood that the drawings are for illustrative purposes only and are not intended to limit the scope of protection of this invention. In the drawings: Figure 1 Retention time calibration curves for standards of different molecular weights; Figure 2 This is a distribution diagram of the molecular weight range of different pancreatic peptide samples; Figure 3 A graph comparing the degree of hydrolysis of different pancreatic peptide samples; Figure 4 Figure showing the effect of compositions obtained by different pancreatic peptide preparation methods on the inhibitory effect on PANC-1 cell proliferation; Figure 5 The figure shows the effect of different animal-derived peptide compositions on the inhibitory effect of PANC-1 cell proliferation. Detailed Implementation

[0020] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments.

[0021] This invention provides an herbal composition containing pancreatic peptides and bone marrow peptides. The composition includes pancreatic peptides, bone marrow peptides, and herbal extracts. The pancreatic peptides are obtained from animal pancreatic tissue after pretreatment, action by endogenous proteases, and further hydrolysis by exogenous proteases.

[0022] The bone marrow peptides were obtained from animal bone marrow tissue through protein extraction, enzymatic hydrolysis, and separation and purification.

[0023] In the preparation of pancreatic peptides, animal pancreatic tissue is first pretreated at low temperature to fully disperse the pancreatic tissue. Then, through self-activation treatment, the endogenous proteases in the pancreatic tissue are gradually restored to their functional state and participate in the protein degradation process. Then, through ethanol-assisted activation and autolysis treatment, the endogenous proteases further act on the pancreatic tissue proteins to form a protein degradation system pretreated with endogenous proteases. Subsequently, exogenous proteases are used to further hydrolyze the above protein degradation system to obtain pancreatic peptides.

[0024] Specifically, animal pancreatic tissue is selected as raw material. Fat, fascia and other non-target tissues on the surface of the pancreatic tissue are removed, and the tissue is washed with water. After washing, the pancreatic tissue is crushed under low temperature to fully disperse the pancreatic tissue and obtain pancreatic tissue slurry.

[0025] In this embodiment, the animal pancreatic tissue is selected from one of pig pancreas, bovine pancreas, and sheep pancreas, preferably pig pancreas. The pancreatic tissue is kept at a low temperature during processing, and the crushing temperature is controlled at 4-10℃ to reduce protein denaturation during processing and maintain the activity of endogenous proteases in the pancreatic tissue.

[0026] The obtained pancreatic tissue slurry was placed at 5-10℃ for self-activation treatment for 24-36 hours, so that the endogenous proteases in the pancreatic tissue could gradually recover their active state and form activated pancreatic tissue slurry.

[0027] Subsequently, ethanol was added to the activated pancreatic tissue slurry for auxiliary activation treatment. The ethanol used was a 25% ethanol solution, and the amount of ethanol solution added was 1-1.5 times the volume of the activated pancreatic tissue slurry. The pH of the system was adjusted to 5-6 to obtain activated pancreatic emulsion.

[0028] The obtained activated pancreatic emulsion was subjected to autolysis treatment, which allowed the endogenous proteases in it to continuously act on the protein substrates in the pancreatic tissue, causing some macromolecular proteins to degrade and forming an autolyzed protein degradation system.

[0029] In this embodiment, during the autolysis process, the pH of the system is controlled at 7.0-7.5, the temperature at 46-48℃, and the treatment time is 8 hours. After the autolysis process is completed, a uniform protein degradation system is formed.

[0030] Subsequently, an exogenous protease is added to the above protein degradation system for further hydrolysis. In this embodiment, the exogenous protease is papain.

[0031] Before adding papain, adjust the pH of the system to 6.0-6.5, add papain at 0.5-2% of the mass of the protein degradation system, and then carry out enzymatic hydrolysis at 58-60℃ for 4 hours.

[0032] After the above treatment, papain acts on the protein substrate that has been pre-degraded by endogenous proteases, causing further hydrolysis of the residual protein to form pancreatic peptide components.

[0033] After enzymatic hydrolysis, the obtained pancreatic peptide hydrolysate is subjected to enzyme inactivation treatment. Specifically, the pancreatic peptide hydrolysate is heated to 85-95℃ and maintained for 10-30 minutes to deactivate the protease in the system.

[0034] After enzyme inactivation, the pancreatic peptide hydrolysate is cooled and then filtered to remove incompletely hydrolyzed protein aggregates and insoluble impurities. The filtered pancreatic peptide solution is then concentrated to obtain pancreatic peptide concentrate.

[0035] Depending on the product form requirements, the pancreatic peptide concentrate can be further dried to obtain pancreatic peptide powder. In this embodiment, spray drying is used to obtain powdered pancreatic peptide.

[0036] Furthermore, bone marrow peptides were prepared using animal bone marrow tissue as raw material.

[0037] Specifically, animal bone marrow tissue is selected, surface impurities are removed, and the tissue is cleaned and then mechanically crushed to fully disperse the bone marrow tissue and obtain bone marrow tissue slurry.

[0038] In this embodiment, the animal bone marrow tissue is selected from bovine bone marrow.

[0039] An extraction solution is added to the obtained bone marrow tissue slurry for protein extraction, allowing the protein components in the bone marrow tissue to enter the extraction system. In this embodiment, the extraction solution is water, and the bone marrow tissue slurry is mixed with water at a mass ratio of 1:8 to 1:12, and extracted at 70-90℃ for 1-3 hours.

[0040] After extraction, the extraction system is filtered to remove bone tissue residue and other insoluble impurities, yielding bone marrow protein extract.

[0041] Subsequently, the obtained bone marrow protein extract was subjected to enzymatic hydrolysis, causing the protein components to degrade and form bone marrow peptides.

[0042] In this embodiment, a staged enzymatic hydrolysis method is adopted. First, alkaline protease is added to the bone marrow protein extract for the first stage of hydrolysis. The amount of alkaline protease added is 0.5-2% of the protein mass in the bone marrow protein extract, so as to cause preliminary degradation of bone marrow protein. Then, the pH of the system is adjusted to 7.5-8.5, and trypsin is added for the second stage of hydrolysis. The amount of trypsin added is 0.3-1% of the protein mass in the bone marrow protein extract, so as to further hydrolyze the protein components after preliminary degradation to form bone marrow peptides.

[0043] The first stage of enzymatic hydrolysis is carried out at a temperature of 45-55℃ for 1-3 hours; the second stage is carried out at a temperature of 35-45℃ for 1-3 hours.

[0044] After enzymatic hydrolysis, the bone marrow peptide hydrolysate is subjected to enzyme inactivation treatment. Specifically, the bone marrow peptide hydrolysate is heated to 85-95℃ and maintained for 10-30 minutes to deactivate the proteases in the system.

[0045] After the enzyme-inactivated bone marrow peptide hydrolysate is cooled and filtered, it is then concentrated and dried to obtain bone marrow peptide powder.

[0046] Furthermore, the herbal raw materials are extracted to obtain herbal extracts.

[0047] The herbal ingredients include ginseng, astragalus, dandelion, Solomon's seal, and saffron, comprising, by weight, 20-40 parts ginseng, 20-40 parts astragalus, 10-30 parts dandelion, 10-30 parts Solomon's seal, and 5-15 parts saffron.

[0048] Specifically, the herbal raw materials are washed, dried, and pulverized to obtain herbal raw material powder, and an extraction solvent is added to the obtained herbal raw material powder for extraction.

[0049] In this embodiment, water is used as the extraction solvent. The herbal raw material powder and water are mixed at a mass ratio of 1:8 to 1:15 and extracted at 80-100℃ for 1-3 hours.

[0050] After extraction, the extraction system is filtered to remove plant residues and obtain herbal extract. The obtained herbal extract is then concentrated to obtain concentrated herbal extract. The concentrated herbal extract can be used directly for composition preparation or further dried to obtain herbal extract powder.

[0051] The pancreatic peptides, bone marrow peptides, and herbal extracts obtained above are mixed to obtain a herbal composition containing pancreatic peptides and bone marrow peptides.

[0052] The components, by mass fraction, include 10-30 parts pancreatic peptide, 10-30 parts bone marrow peptide, and 40-80 parts herbal extract. Preferably, the mass ratio of the pancreatic peptide, bone marrow peptide, and herbal extract is 25:25:50.

[0053] In the specific preparation process, the pancreatic peptide powder, bone marrow peptide powder, and herbal extract powder obtained after drying are weighed according to a predetermined ratio and added to a powder mixing device. The mixture is then mixed at 20-40℃ for 20-60 minutes to fully disperse the components and form a uniform powder mixture, thereby obtaining a solid herbal composition containing pancreatic peptide and bone marrow peptide.

[0054] The present invention also provides specific embodiments of the above-mentioned herbal composition containing pancreatic peptides and bone marrow peptides.

[0055] Example 1 This embodiment provides a herbal composition containing pancreatic peptides and bone marrow peptides, the preparation method of which is as follows: Fresh pig pancreatic tissue was selected, and the surface fat, fascia and other impurities were removed. The tissue was then washed with clean water and crushed at 8°C to obtain pancreatic tissue slurry.

[0056] The obtained pancreatic tissue slurry was treated at 8°C for 30 hours. Then, a 25% ethanol solution was added to the treated pancreatic tissue slurry, with the amount of ethanol solution added being 1.2 times the volume of the pancreatic tissue slurry. The pH of the system was adjusted to 5.5 to obtain the treated pancreatic tissue slurry.

[0057] The pH of the obtained treated pancreatic tissue slurry was adjusted to 7.2 and treated at 47°C for 8 hours. Subsequently, the pH of the system was adjusted to 6.2, and papain was added at 1% of the mass of the protein degradation system. The mixture was then enzymatically hydrolyzed at 59°C for 4 hours to obtain pancreatic peptide hydrolysate.

[0058] The obtained pancreatic peptide hydrolysate was heated to 90°C and held for 20 minutes to inactivate the enzyme. After cooling, it was filtered to remove incompletely hydrolyzed protein aggregates and insoluble impurities. Subsequently, it was concentrated and dried by spray drying to obtain pancreatic peptide powder.

[0059] Bovine bone marrow tissue was collected, surface impurities were removed, and the tissue was cleaned and then mechanically crushed to obtain bone marrow tissue slurry.

[0060] Water was added to the obtained bone marrow tissue slurry for extraction. The bone marrow tissue slurry and water were mixed at a mass ratio of 1:10 and extracted at 80℃ for 2 hours. After extraction, the mixture was filtered to remove bone tissue residue and other insoluble impurities, and bone marrow protein extract was obtained.

[0061] Alkaline protease was added to the obtained bone marrow protein extract for the first stage of hydrolysis. The amount of alkaline protease added was 1% of the protein content in the bone marrow protein extract. The hydrolysis was carried out at 50°C for 2 hours.

[0062] Subsequently, the pH of the system was adjusted to 8.0, and trypsin was added for the second stage of hydrolysis. The amount of trypsin added was 0.5% of the protein content in the bone marrow protein extract, and the enzymatic hydrolysis was carried out at 40°C for 2 hours.

[0063] After enzymatic hydrolysis, the resulting bone marrow peptide hydrolysate is heated to 90°C and kept at that temperature for 20 minutes to inactivate the enzyme. After cooling, filtration, concentration and drying, bone marrow peptide powder is obtained.

[0064] According to the mass fractions, 30 parts of ginseng, 30 parts of astragalus, 20 parts of dandelion, 15 parts of Solomon's seal, and 5 parts of saffron were weighed out respectively. After washing, drying and pulverizing, herbal raw material powder was obtained.

[0065] Water was added to the obtained herbal raw material powder for extraction. The herbal raw material powder and water were mixed at a mass ratio of 1:10 and extracted at 90℃ for 2 hours. After extraction, the mixture was filtered to remove plant residues and obtain herbal extract. The herbal extract was then concentrated and dried to obtain herbal extract powder.

[0066] The obtained pancreatic peptide powder, bone marrow peptide powder, and herbal extract powder were added to a powder mixing device at a mass ratio of 25:25:50 and mixed at 25°C for 30 minutes to ensure thorough mixing of the components, thereby obtaining a herbal composition containing pancreatic peptide and bone marrow peptide.

[0067] Example 2 This embodiment provides a herbal composition containing pancreatic peptides and bone marrow peptides. The difference from Example 1 is that the self-activation treatment conditions during the preparation of pancreatic peptides are different.

[0068] Specifically, fresh pig pancreatic tissue was selected, and the surface fat, fascia and other impurities were removed. The tissue was then washed with clean water and crushed at 6°C to obtain pancreatic tissue slurry.

[0069] The obtained pancreatic tissue slurry was treated at 6°C for 24 hours. Then, a 25% ethanol solution was added to the treated pancreatic tissue slurry, with the amount of ethanol solution added being 1.2 times the volume of the pancreatic tissue slurry. The pH of the system was adjusted to 5.5.

[0070] The pH of the obtained system was adjusted to 7.2 and treated at 47°C for 8 hours. Then, the pH of the system was adjusted to 6.2, and papain was added at 1% of the mass of the protein degradation system. The system was then enzymatically hydrolyzed at 59°C for 4 hours to obtain pancreatic peptide hydrolysate.

[0071] The obtained pancreatic peptide hydrolysate was heated to 90°C and kept at that temperature for 20 minutes to inactivate the enzyme. After cooling, filtration, concentration and spray drying, pancreatic peptide powder was obtained.

[0072] The preparation process of bone marrow peptides and herbal extracts is the same as in Example 1.

[0073] The obtained pancreatic peptide powder, bone marrow peptide powder and herbal extract powder were mixed in a mass ratio of 25:25:50 to obtain a herbal composition.

[0074] Example 3 This embodiment provides a herbal composition containing pancreatic peptides and bone marrow peptides. The difference from Example 1 is that the self-activation treatment conditions during the preparation of pancreatic peptides are different.

[0075] Specifically, fresh pig pancreatic tissue is selected, surface fat, fascia and other impurities are removed, and the tissue is washed with clean water. The washed pancreatic tissue is then crushed at 10°C to obtain pancreatic tissue slurry.

[0076] The obtained pancreatic tissue slurry was treated at 10°C for 36 hours. Then, a 25% ethanol solution was added to the treated pancreatic tissue slurry, with the amount of ethanol solution added being 1.2 times the volume of the pancreatic tissue slurry. The pH of the system was adjusted to 5.5.

[0077] The pH of the obtained system was adjusted to 7.2 and treated at 47°C for 8 hours. Then, the pH of the system was adjusted to 6.2, and papain was added at 1% of the mass of the protein degradation system. The system was then enzymatically hydrolyzed at 59°C for 4 hours to obtain pancreatic peptide hydrolysate.

[0078] The obtained pancreatic peptide hydrolysate was heated to 90°C and kept at that temperature for 20 minutes to inactivate the enzyme. After cooling, filtration, concentration and spray drying, pancreatic peptide powder was obtained.

[0079] The preparation process of bone marrow peptides and herbal extracts is the same as in Example 1.

[0080] The obtained pancreatic peptide powder, bone marrow peptide powder and herbal extract powder were mixed in a mass ratio of 25:25:50 to obtain a herbal composition.

[0081] Comparative Example 1 This comparative example provides a herbal composition containing pancreatic peptides and bone marrow peptides. The difference from Example 1 is that no endogenous protease pretreatment is performed during the preparation of the pancreatic peptides.

[0082] Specifically, the pH of the cleaned and crushed porcine pancreatic tissue slurry was directly adjusted to 6.2, and papain was added at 1% of the mass of the protein degradation system. The mixture was then enzymatically hydrolyzed at 59°C for 4 hours to obtain pancreatic peptide hydrolysate.

[0083] The obtained pancreatic peptide hydrolysate was heated to 90°C and kept at that temperature for 20 minutes to inactivate the enzyme. After cooling, filtration, concentration and spray drying, pancreatic peptide powder was obtained.

[0084] Subsequently, the obtained pancreatic peptide powder was mixed with the bone marrow peptide powder prepared in Example 1 and the herbal extract powder at a mass ratio of 25:25:50 to obtain the composition.

[0085] Comparative Example 2 This comparative example provides a herbal composition containing pancreatic peptides and bone marrow peptides. The difference from Example 1 is that no exogenous protease is added for further hydrolysis during the preparation of the pancreatic peptides.

[0086] Specifically, fresh porcine pancreatic tissue was selected and processed according to the steps of low-temperature disruption, self-activation, ethanol-assisted activation, and autolysis in Example 1 to obtain a protein degradation system.

[0087] After processing, without adding papain, the resulting protein degradation system was directly heated to 90°C and kept at that temperature for 20 minutes to inactivate the enzyme. After cooling, filtration, concentration and spray drying, pancreatic peptide powder was obtained.

[0088] Subsequently, the obtained pancreatic peptide powder was mixed with the bone marrow peptide powder prepared in Example 1 and the herbal extract powder at a mass ratio of 25:25:50 to obtain the composition.

[0089] Comparative Example 3 This comparative example provides a herbal composition that differs from Example 1 in that it does not contain pancreatic peptides.

[0090] Specifically, the bone marrow peptide powder obtained in Example 1 and the herbal extract powder were mixed at a mass ratio of 25:75 to obtain the composition.

[0091] Comparative Example 4 This comparative example provides a herbal composition that differs from Example 1 in that it does not contain bone marrow peptides.

[0092] Specifically, the pancreatic peptide powder obtained in Example 1 and the herbal extract powder were mixed at a mass ratio of 25:75 to obtain the composition.

[0093] Comparative Example 5 This comparative example provides a herbal composition containing animal-derived peptides, which differs from Example 1 in that it uses animal-derived peptides that have not been pretreated with endogenous proteases instead of the pancreatic peptides in Example 1.

[0094] Specifically, after cleaning and crushing the pig pancreatic tissue, the pH of the system was directly adjusted to 6.2, and papain was added at 1% of the mass of the protein degradation system. The mixture was then enzymatically hydrolyzed at 59°C for 4 hours to obtain animal-derived peptide powder.

[0095] The obtained animal-derived peptide powder was mixed with the bone marrow peptide powder prepared in Example 1 and the herbal extract powder at a mass ratio of 25:25:50 to obtain the composition.

[0096] To verify the compositional characteristics and application effects of the compositions obtained in this invention, experimental examples are provided below for further explanation.

[0097] Experimental Example 1 Pancreatic peptide composition status detection The pancreatic peptide powders prepared in Example 1, Comparative Example 1, and Comparative Example 2 were used as test samples to detect their molecular weight distribution and degree of hydrolysis.

[0098] Weigh 0.5g of the pancreatic peptide powder samples prepared in Example 1, Comparative Example 1 and Comparative Example 2 respectively, add deionized water to prepare a test solution with a mass concentration of 1mg / mL, centrifuge after full dissolution, and filter the supernatant through a 0.22μm filter membrane to obtain the test sample solution.

[0099] The obtained sample solution was detected by high performance gel permeation chromatography. The chromatographic column was a TSK gel G2000SWXL gel column. The mobile phase was a mixed solution of acetonitrile and water with a volume ratio of 30:70. 0.1% trifluoroacetic acid was added to the mobile phase. The flow rate was 0.5 mL / min, the detection wavelength was 220 nm, and the injection volume was 10 μL.

[0100] A molecular weight calibration curve for gel chromatography was established. Specifically, blue dextran was used as the size exclusion volume marker for the gel chromatography column, and bovine serum albumin, ovalbumin, cytochrome C, aprotinin, bacitracin, and glutathione were used as molecular weight calibration standards. The molecular weights of the standards were 66,000 Da, 43,000 Da, 12,400 Da, 6,500 Da, 1,422 Da, and 307 Da, respectively.

[0101] By determining the retention times of various molecular weight standards during gel chromatography analysis, a correlation between retention time and the logarithmic value of molecular weight was established, resulting in a molecular weight calibration curve. The corresponding molecular weights were calculated based on the retention times of each chromatographic peak in the pancreatic peptide sample, and the relative content of peptide components in different molecular weight ranges was analyzed using peak area integral results. Figure 1 As shown.

[0102] Figure 1 To obtain calibration curves for the retention times of standards with different molecular weights, the retention times of different molecular weight standards during gel chromatography analysis were measured, and the correspondence between retention time and logarithmic molecular weight was established, thus obtaining calibration curves for the molecular weight analysis of peptide components in samples. Figure 1 It can be seen that as the molecular weight of the standard decreases, its retention time in the gel chromatography analysis gradually increases, and the resulting calibration curve has a good linear relationship, which can be used for subsequent calculation and analysis of the molecular weight range distribution of pancreatic peptide samples.

[0103] The above-mentioned sample solutions were taken separately, and the free amino content was determined by the o-phthalaldehyde method (OPA method). A standard curve was established using L-leucine as a standard. The absorbance was measured at a wavelength of 340 nm. The degree of hydrolysis of the sample was calculated based on the free amino content. Three parallel samples were set up for each of the above tests. The results are shown in Table 1. Figure 2 as well as Figure 3 .

[0104] Table 1. Results of compositional analysis of different pancreatic peptide samples

[0105] From Table 1 and Figure 2 , Figure 3 It can be seen that the pancreatic peptides obtained in Example 1 have the highest content of small molecule peptides with a molecular weight of <1000Da and the lowest content of large molecule peptides with a molecular weight of >3000Da. At the same time, the degree of hydrolysis is also higher than that of Comparative Example 1 and Comparative Example 2. This indicates that the method of using endogenous protease pretreatment combined with exogenous protease for further hydrolysis is beneficial to promote the full degradation of pancreatic tissue proteins and make the obtained pancreatic peptides form a more reasonable molecular weight distribution.

[0106] Experimental Example 2 To further verify the effect of differences in the pancreatic peptide formation process on the functional effects of the composition, herbal compositions containing pancreatic peptides and bone marrow peptides prepared in Example 1, Comparative Example 1, and Comparative Example 2 were selected as test samples.

[0107] Weigh the above-mentioned composition samples separately, add sterile PBS buffer to dissolve them, and prepare a sample stock solution with a mass concentration of 10 mg / mL. After thorough mixing, centrifuge the solution and take the supernatant as the test sample solution. Further dilute the obtained test sample solution to obtain test samples with mass concentrations of 25 μg / mL, 50 μg / mL and 100 μg / mL.

[0108] The human pancreatic cancer cell line PANC-1 was used as the detection target. PANC-1 is a publicly available human pancreatic cancer cell line that can be obtained through ATCC and has the corresponding registration number CRL-1469.

[0109] PANC-1 cells in the logarithmic growth phase were seeded into 96-well culture plates at a density of 5 × 10⁶ cells per well. 3 Each sample was cultured in DMEM medium containing 10% fetal bovine serum at 37°C with 5% carbon dioxide.

[0110] After the cells adhered to the cell wall, different concentrations of the test sample solution were added. Cells without the added sample solution were used as a blank control group. The cells were cultured for another 48 hours. After the culture was completed, CCK-8 test reagent was added to each well and the cells were incubated for another 2 hours. The absorbance at 450 nm was measured using an ELISA reader.

[0111] The cell proliferation inhibition rate was calculated based on the detected absorbance values. The cell proliferation inhibition rate was defined as the percentage decrease in absorbance value of the experimental group treated with the sample compared to the blank control group without sample treatment. All experiments used three parallel wells. The results are shown in Table 2. Figure 4 .

[0112] Table 2. Effects of different pancreatic peptide-derived compositions on PANC-1 cell proliferation.

[0113] From Table 2 and Figure 4 It can be seen that as the concentration of the test sample increases, the inhibitory effect of each composition on the proliferation of PANC-1 cells gradually increases. Under the same detection concentration conditions, the cell proliferation inhibition rate of the composition obtained in Example 1 is higher than that of Comparative Example 1 and Comparative Example 2.

[0114] Based on the test results of Example 1, it can be seen that Example 1 uses endogenous protease pretreatment combined with exogenous protease for further hydrolysis, so that the pancreatic peptides obtained have a different compositional state from those obtained by direct exogenous enzyme hydrolysis, and further improve the inhibitory effect of the composition on PANC-1 cell proliferation. Comparative Example 1 did not undergo endogenous protease pretreatment, and Comparative Example 2 did not undergo exogenous protease for further hydrolysis, so the pancreatic peptides obtained changed their formation state, resulting in a decrease in the overall functional effect of the composition.

[0115] The results showed that the formation process of pancreatic peptides affects their effect in the composite composition. This invention improves the composition of the obtained pancreatic peptides by continuously combining the endogenous protease action stage and the exogenous protease hydrolysis stage, and enhances the inhibitory effect of the herbal composition containing pancreatic peptides and bone marrow peptides on the proliferation of pancreatic cancer cells.

[0116] Experimental Example 3 To verify the effect of different combinations of pancreatic peptides, bone marrow peptides and herbal extracts on the functional effects of the composition, the compositions prepared in Example 1, Comparative Example 3 and Comparative Example 4 were used as test samples.

[0117] Weigh the above-mentioned composition samples separately, and dissolve and dilute them according to the sample processing method in Experimental Example 2 to obtain a test sample solution with a mass concentration of 100 μg / mL.

[0118] Using the cell culture conditions and CCK-8 assay method described in Experiment 2, the effects of different combinations on the proliferation of PANC-1 cells were investigated. Three parallel wells were set up for each experiment, and the results are shown in Table 3.

[0119] Table 3. Effects of different component combinations on PANC-1 cell proliferation.

[0120] As shown in Table 3, under the same detection concentration conditions, the cell proliferation inhibition rate of the composition obtained in Example 1 was higher than that of Comparative Examples 3 and 4, indicating that the composition can exhibit a better inhibitory effect when pancreatic peptides, bone marrow peptides and herbal extracts are present together.

[0121] The cell proliferation inhibition effect of Comparative Example 3 was significantly reduced, indicating that the pancreatic peptide obtained through a specific process is an important component affecting the functional effect of the composition. Although Comparative Example 4 still contains pancreatic peptide and herbal extract, its cell proliferation inhibition effect is still lower than that of Example 1, indicating that there is a combination relationship between bone marrow peptide, pancreatic peptide and herbal extract, and the absence of any animal-derived peptide component will affect the overall effect of the composition.

[0122] The results show that the functional effects of the composition of the present invention do not originate from a single component, but are produced by a complex system composed of pancreatic peptides, bone marrow peptides and herbal extracts.

[0123] Experiment Example 4 To verify the functional difference between the pancreatic peptide obtained in this invention and the pancreatic peptide obtained without endogenous protease pretreatment in the composition, the compositions prepared in Example 1 and Comparative Example 5 were used as test samples.

[0124] Weigh the above-mentioned composition samples separately, add sterile PBS buffer to dissolve them, and prepare a sample stock solution with a mass concentration of 10 mg / mL. After thorough mixing, centrifuge the solution and take the supernatant as the test sample solution. Further dilute the obtained test sample solution to obtain test samples with mass concentrations of 25 μg / mL, 50 μg / mL and 100 μg / mL.

[0125] Human pancreatic cancer cells PANC-1 were used as the detection target and cultured according to the cell culture conditions in Experiment 2.

[0126] After cell adhesion, different concentrations of the test sample solution were added, with cells without the sample solution serving as a blank control group. Cells were cultured for another 48 hours. After culture, cell proliferation was measured using the CCK-8 assay, and the cell proliferation inhibition rate was calculated based on the absorbance values. The experiment was conducted with three parallel wells. The results are shown in Table 4. Figure 5 .

[0127] Table 4. Effects of different animal-derived peptide sources on the functional effects of the composition.

[0128] From Table 4 and Figure 5 It can be seen that, under the same detection concentration conditions, the inhibitory effect of the composition obtained in Example 1 on the proliferation of PANC-1 cells is higher than that in Comparative Example 5, and the cell proliferation inhibition effect of both compositions gradually increases with the increase of detection concentration.

[0129] Comparative Example 5 used common animal-derived peptides instead of pancreatic peptides in Example 1. While maintaining the same content of animal-derived peptides and the same proportion of other components in the composition, its cell proliferation inhibition effect was significantly reduced, indicating that the formation mode of animal-derived peptides affects their effect in the composite composition.

[0130] Based on the test results of Experiment 1 and Experiment 2, it can be seen that the present invention combines the endogenous protease action stage with the exogenous protease further hydrolysis stage, so that the resulting pancreatic peptides form a compositional state that is different from ordinary animal-derived peptides, and further improves the inhibitory effect of the herbal composition containing pancreatic peptides and bone marrow peptides on the proliferation of pancreatic cancer cells.

[0131] The technical scope of this invention is not limited to the content described above. Those skilled in the art can make various modifications and variations to the above embodiments without departing from the technical concept of this invention, and all such modifications and variations should fall within the protection scope of this invention.

Claims

1. A herbal composition containing pancreatic peptides and bone marrow peptides, characterized in that, It includes pancreatic peptides, bone marrow peptides, and herbal extracts, which are obtained by extraction and processing of ginseng, astragalus, dandelion, Solomon's seal, and saffron; The pancreatic peptides were obtained by cryogenically disrupting animal pancreatic tissue at 4-10°C to obtain pancreatic tissue slurry. The obtained pancreatic tissue slurry was then treated at 5-10°C for 24-36 hours to allow the endogenous protease system contained in the animal pancreatic tissue to gradually participate in protein degradation. Subsequently, a 25% (v / v) ethanol solution was added for auxiliary activation treatment. The amount of ethanol solution added was 1-1.5 times the volume of the pancreatic tissue slurry, and the pH of the system was adjusted to 5-6, and then adjusted to 7.0-7.

5. The system was then autolyzed at 46-48°C for 8 hours to form a protein degradation system. The protein degradation system was then hydrolyzed using papain to obtain the final product.

2. The herbal composition containing pancreatic peptides and bone marrow peptides according to claim 1, characterized in that: Based on weight parts, it includes 10-30 parts pancreatic peptides, 10-30 parts bone marrow peptides, and 40-80 parts herbal extracts.

3. The herbal composition containing pancreatic peptides and bone marrow peptides according to claim 1 or 2, characterized in that: The raw materials for preparing the herbal extract, by weight, include 20-40 parts ginseng, 20-40 parts astragalus, 10-30 parts dandelion, 10-30 parts Solomon's seal and 5-15 parts saffron.

4. The herbal composition containing pancreatic peptides and bone marrow peptides according to claim 1, characterized in that: The animal pancreatic tissue is selected from one of the following: porcine pancreas, bovine pancreas, or sheep pancreas. The bone marrow peptide is obtained from animal bone marrow tissue through protein extraction, enzymatic hydrolysis, and separation and purification.

5. The herbal composition containing pancreatic peptides and bone marrow peptides according to claim 1 or 2, characterized in that: The mass ratio of the pancreatic peptide, bone marrow peptide, and herbal extract is 25:25:

50.

6. A method for preparing a herbal composition containing pancreatic peptides and bone marrow peptides as described in any one of claims 1-5, characterized in that, Includes the following steps: S1: Using animal pancreatic tissue as raw material, the pancreatic tissue is subjected to low-temperature crushing at 4-10℃ to obtain pancreatic tissue slurry. The obtained pancreatic tissue slurry is then treated at 5-10℃ for 24-36 hours to allow the endogenous protease system contained in the animal pancreatic tissue to gradually participate in protein degradation. Subsequently, a 25% (v / v) ethanol solution is added for auxiliary activation treatment. The amount of ethanol solution added is 1-1.5 times the volume of the pancreatic tissue slurry. The pH of the system is adjusted to 5-6, and then adjusted to 7.0-7.

5. Autolysis is performed at 46-48℃ for 8 hours to form a protein degradation system. The pH of the system is then adjusted to 6.0-6.5, and papain is added at 0.5-2% of the mass of the protein degradation system. Enzymatic hydrolysis is performed at 58-60℃ for 4 hours to obtain pancreatic peptides. S2: Bone marrow peptides are obtained from animal bone marrow tissue through protein extraction, enzymatic hydrolysis, and separation and purification. S3: Based on the mass fractions, take 20-40 parts of ginseng, 20-40 parts of astragalus, 10-30 parts of dandelion, 10-30 parts of Solomon's seal and 5-15 parts of saffron for extraction to obtain herbal extracts. S4: Mix the obtained pancreatic peptides, bone marrow peptides and herbal extracts to obtain a herbal composition containing pancreatic peptides and bone marrow peptides.

7. The method for preparing the herbal composition containing pancreatic peptides and bone marrow peptides according to claim 6, characterized in that: In step S1, after the papain is digested, the resulting pancreatic peptide hydrolysate is heated to 85-95℃ and kept at that temperature for 10-30 minutes to inactivate the enzyme. After cooling, filtration and concentration, a concentrated pancreatic peptide solution is obtained.

8. The method for preparing the herbal composition containing pancreatic peptides and bone marrow peptides according to claim 6, characterized in that: In step S3, the herbal raw materials are washed, dried and crushed, and then mixed with water at a mass ratio of 1:8 to 1:

15. The mixture is extracted at 80-100℃ for 1-3 hours, and the herbal extract is obtained by filtration and concentration.

9. The method for preparing the herbal composition containing pancreatic peptides and bone marrow peptides according to claim 6, characterized in that: The animal bone marrow tissue is bovine bone marrow. The animal bone marrow tissue is extracted with water to obtain bone marrow protein extract, which is then processed by a staged enzymatic hydrolysis method, including adding alkaline protease for the first stage of hydrolysis, and adjusting the pH of the system to 7.5-8.5 before adding trypsin for the second stage of hydrolysis.

10. The use of an herbal composition containing pancreatic peptides and bone marrow peptides as described in any one of claims 1-5 in the preparation of a product for inhibiting the proliferation of pancreatic cancer cells.

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