Echinacea micromolecule peptide composition for promoting yield increase of natural calculus bovis

Abstract

The invention discloses an echinacea micromolecule peptide composition for promoting yield increase of natural calculus bovis. The echinacea micromolecule peptide composition comprises four sections of micromolecule peptides of peptide sequences A, B, C and D, the composition is extracted from echinacea through a specific process, and the proportion of each peptide sequence is 15-35%. When the sodium alginate microsphere carrier is prepared into a solution to be injected or loaded on the sodium alginate microsphere carrier to be implanted near the gallbladder of cattle, the formation rate of the natural calculus bovis can be remarkably improved to 60-65%, the average weight is greatly increased, and cholecystitis can be relieved. Compared with a traditional method, the technology is safe, efficient, wide in raw material source, low in cost and capable of specifically acting on bezoar to form a key target spot, and bezoar formation is promoted through cooperation of multiple links.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and specifically relates to a technique for extracting a small molecule peptide sequence composition from Echinacea purpurea for promoting the production of natural bezoar and its application. Background Art

[0002] As a gem in traditional Chinese medicines, natural bezoar has extremely important medicinal value. It has many effects such as clearing the heart, resolving phlegm, opening orifices, cooling the liver, calming endogenous wind, and detoxifying. It performs outstandingly in treating various difficult and complicated diseases. For example, in Angong Niuhuang Pills, natural bezoar plays a key role and has significant curative effects on severe diseases such as coma due to febrile diseases; Niuhuang Jiedu Tablets, with it as the main ingredient, has good effects in relieving symptoms such as sore throat and sores in the mouth.

[0003] With the wide spread of traditional Chinese medicine globally and the continuous increase in people's attention to health, the market demand for natural bezoar has shown a blowout growth. However, the natural formation probability of natural bezoar is extremely low. According to statistics, only about 1 - 2 cows out of every 100,000 cows can produce natural bezoar, and the output is extremely scarce, which has caused its price to soar continuously, and the market has been in a long-term tight state of supply falling short of demand. The high price has greatly increased the production cost of Chinese patent medicines containing natural bezoar, severely restricting the clinical application and market promotion of these drugs.

[0004] Currently, there are obvious deficiencies in the production technology of natural bezoar. Although the foreign body implantation method in the gallbladder attempts to stimulate the formation of bezoar by implanting foreign bodies into the bovine gallbladder, this method is extremely likely to cause serious complications such as bovine gallbladder infection and inflammation, which will not only lead to a sharp deterioration in the health of cows but may even cause the death of cows. Moreover, the bezoar produced by this method has uneven quality and large differences in the content of active ingredients, and it is difficult to meet the strict medicinal standards of natural bezoar. Although the in vitro cultivation method can simulate the internal environment of cows to a certain extent to cultivate bezoar and has a certain control ability over the quality of bezoar, this technology has extremely high requirements for equipment and technology, requires a huge upfront investment in equipment, has high production costs, and has a long cultivation cycle, and cannot meet the growing large-scale production demand.

[0005] Therefore, it is extremely urgent to develop a highly efficient, safe and low-cost technology for promoting the production of natural bezoar, which has important practical significance for meeting market demand and promoting the development of the traditional Chinese medicine industry. Summary of the Invention

[0006] The present invention provides a small molecule peptide composition for promoting the production of natural bezoar, comprising the following small molecule peptide sequences:

[0007] Peptide sequence A: Ala - Asp - Lys - Thr - Gly - Ser - Pro - Tyr - Leu - Ile - Val - Phe - Met - His;

[0008] Peptide sequence B: Gly-Gln-Asn-Arg-Pro-Thr-Ser-Ile-Leu-Phe-Tyr-Trp-Lys-Glu;

[0009] Peptide sequence C: Thr-Lys-Glu-Asp-Pro-Gly-Ser-Val-Ile-Leu-Phe-Tyr-Trp-His-Ala-Arg;

[0010] Peptide sequence D: Pro-Arg-Glu-Asp-Gly-Ser-Thr-Ile-Val-Leu-Phe-Tyr-Trp-Asn-Gln-Lys;

[0011] The small molecule peptide sequence is derived from the extract of Echinacea.

[0012] Furthermore, the content ratio of each peptide sequence in the small molecule peptide composition is as follows: peptide sequence A accounts for 15%-35%, peptide sequence B accounts for 15%-35%, peptide sequence C accounts for 15%-35%, and peptide sequence D accounts for 15%-35%.

[0013] Furthermore, the preparation method of the small molecule peptide composition is characterized by including the following steps: collecting the above-ground part of Echinacea with strong growth and no diseases and pests, thoroughly rinsing it with clean water to remove surface soil, impurities and microorganisms, and cutting it into small sections of 1-3 cm;

[0014] Using vacuum freeze-drying technology, drying the small sections of Echinacea at -35°C to -25°C until the water content is below 7%, and crushing them into a coarse powder that can pass through a 70-90 mesh sieve;

[0015] Using organic solvents such as acetone, butanol, chloroform and mixed solvents with different ratios, changing the extraction temperature (30°C - 50°C), time (12 - 28 hours), and solid-liquid ratio (1:5 (g / mL) - 1:9 (g / mL)) to conduct extraction experiments on the coarse powder, collecting the extract by centrifugation or filtration, and concentrating it under reduced pressure to obtain an extract concentrate;

[0016] Preliminarily separating the extract concentrate through a macroporous adsorption resin column, selecting a suitable macroporous adsorption resin according to the polarity and molecular size of peptide components, eluting with different concentrations of acetone solution in a gradient manner, and collecting the eluate that may contain peptide components;

[0017] Further separating the above eluate by gel filtration chromatography, selecting gel media such as Sephadex G-50 and Sephacryl S-100, separating according to the difference in peptide molecular size, and collecting the eluate at different time periods for preliminary grouping;

[0018] The peptide components after grouping are further separated by ion exchange chromatography. Cation exchange resin CM-Sepharose or anion exchange resin DEAE-Sepharose, etc. are selected according to the charge properties and quantities of peptide molecules. The pH value and ionic strength of the eluent are adjusted, and the solutions of different elution peaks are collected to obtain relatively purified peptide components;

[0019] The relatively purified peptide components are sequenced by Edman degradation method or mass spectrometry sequencing method. Peptide sequences A, B, C, and D are determined through bioinformatics analysis, and a small molecule peptide composition is obtained by mixing.

[0020] Furthermore, in the preliminary separation step on the macroporous adsorption resin column, macroporous adsorption resins with high polarity are selected for peptide components with high polarity, and macroporous adsorption resins with non-polar or weak polarity are selected for non-polar or weakly polar peptide components.

[0021] Furthermore, in the mass spectrometry sequencing method, the peptide samples are first pretreated such as desalting and concentration. After being injected into the mass spectrometer, ionization methods such as electrospray ionization ESI or matrix-assisted laser desorption ionization MALDI are selected, and professional software is used to analyze the mass spectrometry data.

[0022] Furthermore, the application of the said small molecule peptide composition in promoting the production of natural bezoar.

[0023] Furthermore, the said small molecule peptide composition is formulated into a solution and administered to cattle by intravenous injection at a dose of 3 mg / kg body weight, twice a week for 10 consecutive weeks.

[0024] Furthermore, a method for enhancing the efficiency of promoting the production of natural bezoar, characterized in that sodium alginate with good biocompatibility and biodegradability is selected to prepare microsphere carriers with a diameter of 1-2 mm. The said small molecule peptide sequences are respectively or mixedly loaded onto the microsphere carriers. The microsphere carriers loaded with small molecule peptide sequences or compositions are implanted near the gallbladder of cattle through surgery. After the surgery, the experimental cattle are carefully nursed. When necessary, the small molecule peptides obtained by extraction and purification are formulated into a solution and administered as an adjuvant by intravenous injection at a dose of 3 mg / kg body weight, twice a week for 10 consecutive weeks.

[0025] Furthermore, the loading of small molecule peptide sequences onto the microsphere carriers adopts physical adsorption or chemical coupling methods. In the physical adsorption method, the microsphere carriers and the small molecule peptide solution are mixed for a certain time under appropriate temperature and stirring conditions. In the chemical coupling method, covalent connection is achieved through chemical reactions between the surface active groups of the carrier and the specific functional groups of the small molecule peptides.

[0026] Furthermore, the said pharmaceutical preparation also contains pharmaceutically acceptable carriers, excipients or diluents, and the dosage form is injection, capsule, tablet or granule.

[0027] Beneficial effects

[0028] High efficiency: It can significantly increase the formation rate and yield of natural bezoar. The experimental results show that after using the small molecule peptide sequence and composition of the present invention, the formation rate of bezoar has increased from 20% in the control group to 60%-65%, and the average weight has also increased significantly. Especially, the effect of the small molecule peptide composition group is more prominent.

[0029] Safety: Small molecule peptides extracted from plants are used and combined with a biodegradable carrier with good biocompatibility, which has no obvious adverse effects on the health of cows and avoids serious complications such as infection and inflammation that may be caused by traditional methods such as gallbladder implantation of foreign objects. The carrier gradually degrades in the body and will not cause long-term potential harm to animals.

[0030] Specificity: Through target design, small molecule peptides can specifically act on key targets related to the formation of bezoar, such as enzymes, receptors, etc., improving the pertinence and effectiveness of the action. Different small molecule peptide sequences play roles in different links such as promoting bile acid synthesis, bilirubin uptake, and gallbladder contraction, and cooperate with each other to comprehensively promote the formation of bezoar.

[0031] Cost-effectiveness: The extraction process is relatively simple, the raw material source of Echinacea is wide, and the production cost is low. The price of the carrier material is relatively low, and the preparation process is not complicated, with good economic benefits and market application prospects. Compared with the in vitro cultivation method, it does not require high equipment investment and complex technical operations, reducing the production threshold. Specific implementation methods

[0032] Example 1

[0033] Raw material collection and processing: Carefully select Echinacea plants that are growing vigorously and free of pests and diseases. Collect their above-ground parts, ensuring the integrity of the plants during the collection process. Quickly and thoroughly rinse the collected Echinacea with clean water to remove the attached soil, impurities, and various microorganisms on the surface. After washing, cut it into small sections about 1-3 cm in length for subsequent processing.

[0034] Drying and pulverization: The vacuum freeze-drying technique is used to dry small segments of Echinacea in a low-temperature environment of -35°C to -25°C, reducing the water content to below 7%. This drying method can maximize the retention of bioactive components in Echinacea. The dried Echinacea is processed by a pulverization device to form a coarse powder, and its particle size is controlled to pass through a 70-90 mesh sieve, ensuring that the coarse powder can fully contact the extraction solvent during the subsequent extraction process and improving the extraction efficiency. Optimization of the extraction process: Extraction experiments are conducted on the Echinacea coarse powder using various organic solvents (such as acetone, butanol, chloroform, etc.) and mixed solvents with different ratios. By changing key conditions such as extraction temperature, time, and solid-liquid ratio, multiple groups of parallel experiments are designed. For example, the acetone concentrations are set at 30%, 40%, 50%, 60%, and 70% respectively, the solid-liquid ratio ranges from 1:5 (g / mL) to 1:9 (g / mL), the extraction time ranges from 12 hours to 28 hours, and the extraction temperature ranges from 30°C to 50°C for different combinations. After extraction, the extract is collected by centrifugation or filtration and concentrated under reduced pressure using equipment such as a rotary evaporator to obtain the concentrated Echinacea extract under different conditions. Establishment of an in vitro cell model: Based on liver cells, a cell culture system is constructed that can accurately simulate processes closely related to the formation of bezoar, such as bile acid synthesis and bilirubin uptake. The concentrated Echinacea extracts obtained under different conditions are respectively applied to this cell model. When studying the activity related to bile acid synthesis, the activity change of cholesterol 7α-hydroxylase (CYP7A1) in the cells is mainly detected; when studying the activity related to bilirubin uptake, key indicators such as the change in the bilirubin uptake amount by the cells are carefully observed. By comparing the differences in cell-related indicators after the action of different extracts, the extracts with potential activity in promoting the formation of bezoar are screened out.

[0035] Animal pre-experiment: A small number of healthy experimental animals (such as mice or rats) are selected, and the screened Echinacea extract with potential activity is administered to the animals through appropriate administration routes (such as intraperitoneal injection, gavage, etc.). During the administration period, the physiological state and behavioral changes of the animals are closely observed to ensure that the extract has no obvious toxicity. At the same time, samples such as blood and bile of the animals are regularly collected to detect biochemical indicators related to the formation of bezoar, such as bile acid content and bilirubin level. Based on the results of the animal pre-experiment, the Echinacea extract with significant activity in promoting the formation of bezoar is further determined.

[0036] Preliminary separation by macroporous adsorption resin column: For the Echinacea purpurea extract determined by activity screening, a macroporous adsorption resin column is used for preliminary separation. According to different polarities and molecular sizes, a suitable type of macroporous adsorption resin is accurately selected. For peptide components with relatively high polarity, a polar macroporous adsorption resin is selected; for non-polar or weakly polar peptide components, a non-polar macroporous adsorption resin is chosen. Gradient elution is carried out with acetone solutions of different concentrations, and the solutions of different elution peaks are collected to preliminarily separate the eluate that may contain peptide components.

[0037] Separation by gel filtration chromatography: The eluate that may contain peptide components obtained from the preliminary separation is further separated by gel filtration chromatography. A suitable gel medium (such as Sephadex G-50, Sephacryl S-100, etc.) is selected to separate according to the size differences of peptide molecules. Smaller peptide molecules will diffuse in the pores of the gel particles and have a longer elution time; larger peptide molecules cannot enter the pores of the gel particles and have a shorter elution time. By collecting the eluates at different time intervals, the peptide components are preliminarily grouped according to molecular size.

[0038] Further separation by ion exchange chromatography: The different groups of peptide components obtained from the gel filtration chromatography separation are further separated by ion exchange chromatography. According to the nature and quantity of the charges carried by peptide molecules, a suitable ion exchange medium (such as cation exchange resin CM-Sepharose, anion exchange resin DEAE-Sepharose, etc.) is selected. By adjusting the pH value and ionic strength of the eluate, peptide components with different charge natures and quantities are separated on the ion exchange column, and the solutions corresponding to different elution peaks are collected to obtain relatively purified peptide components.

[0039] Application of sequencing technology: For the relatively purified peptide components obtained through the above separation steps, advanced sequencing technology is used for sequence determination. Currently, commonly used peptide sequencing technologies include Edman degradation method and mass spectrometry sequencing method. The Edman degradation method determines the peptide sequence by gradually degrading and identifying the amino acid residues at the N-terminus of the peptide chain in sequence. The mass spectrometry sequencing method ionizes peptide molecules using a mass spectrometer and infers the peptide sequence by analyzing information such as the mass-to-charge ratio of the ions. Mass spectrometry sequencing process: When using the mass spectrometry sequencing method, first, the peptide sample is pretreated to make it suitable for mass spectrometry analysis. For example, operations such as desalting and concentration are carried out to improve the purity and concentration of the sample. Then the treated sample is injected into the mass spectrometer, and a suitable ionization method (such as electrospray ionization ESI or matrix-assisted laser desorption ionization MALDI, etc.) is selected. After obtaining the mass spectrometry diagram, professional data analysis software is used to analyze the mass spectrometry data. By analyzing information such as the parent ion mass and fragment ion mass of the peptide segment, combined with the known amino acid mass database, the amino acid sequence of the peptide molecule is inferred.

[0040] Bioinformatics analysis: Perform bioinformatics analysis on the peptide sequences obtained by sequencing. By comparing with known protein and peptide sequence databases (such as the protein database of NCBI, etc.), determine whether it is a new sequence. At the same time, analyze the structural characteristics of the peptide sequences, such as amino acid composition, hydrophilicity / hydrophobicity, secondary structure prediction, etc., to provide a basis for subsequent studies on their functions and action mechanisms. After a large amount of screening and sequencing work, the following 4 unreported small molecule peptide sequences were finally determined:

[0041] Peptide sequence A: Ala - Asp - Lys - Thr - Gly - Ser - Pro - Tyr - Leu - Ile - Val - Phe - Met - His

[0042] Peptide sequence B: Gly - Gln - Asn - Arg - Pro - Thr - Ser - Ile - Leu - Phe - Tyr - Trp - Lys - Glu

[0043] Peptide sequence C: Thr - Lys - Glu - Asp - Pro - Gly - Ser - Val - Ile - Leu - Phe - Tyr - Trp - His - Ala - Arg

[0044] Peptide sequence D: Pro - Arg - Glu - Asp - Gly - Ser - Thr - Ile - Val - Leu - Phe - Tyr - Trp - Asn - Gln - Lys

[0045] Vector selection and preparation: Select sodium alginate with good biocompatibility and biodegradability, and prepare microsphere vectors with a diameter of about 1 - 2 mm. Load the above small molecule peptide sequences separately or in combination onto the microsphere vectors. The loading process uses physical adsorption or chemical coupling methods to ensure that the small molecule peptides can be stably bound to the surface or inside of the vectors. For example, for the physical adsorption method, the microsphere vectors can be mixed with the small molecule peptide solution for a certain time under appropriate temperature and stirring conditions to allow the small molecule peptides to adsorb onto the vector surface; for the chemical coupling method, chemical reactions can be carried out between the active groups on the vector surface and the specific functional groups of the small molecule peptides to achieve covalent connection.

[0046] Grouping of experimental animals: Select 80 healthy Simmental cattle with a body weight between 400 - 500 kg, and randomly divide them into 6 groups, with 10 animals in each group. They are the control group, the vector control group, the peptide sequence A group, the peptide sequence B group, the peptide sequence C group, the peptide sequence D group, and the small molecule peptide combination (A + B + C + D) group.

[0047] Experimental methods:

[0048] Carrier control group: Microspheres without small molecule peptides were surgically implanted near the gallbladder of cows, and the surgical procedure strictly followed the aseptic operation specifications.

[0049] Peptide sequence group and composition group: Microspheres loaded with corresponding small molecule peptide sequences or small molecule peptide compositions were also surgically implanted near the gallbladder of cows. After the surgery, all experimental cows were carefully cared for to ensure their recovery to normal physiological states.

[0050] Drug administration supplement (if necessary): To further enhance the effect, for the peptide sequence group and composition group, after the carrier implantation, the small molecule peptides obtained by extraction and purification can also be formulated into a solution and administered as an adjuvant by intravenous injection at a dose of 3 mg / kg body weight, twice a week for 10 consecutive weeks. The control group and the carrier control group were injected with an equal amount of normal saline. Sample collection and detection:

[0051] During the experiment, blood and bile samples were collected every 3 weeks to detect the activities of related enzymes in the blood and the contents of components such as bile acids and bilirubin in the bile.

[0052] After the experiment, the experimental cows were slaughtered, the gallbladders were removed, the formation of bezoar was carefully observed, the weight and size of the bezoar were accurately measured, and a comprehensive chemical composition analysis was carried out.

[0053] Part of the gallbladder tissue was taken for pathological section examination to observe the inflammation of the gallbladder tissue and the compatibility between the carrier and the tissue.

[0054] Peptide sequence A group: After 3 weeks of drug administration, the activity of CYP7A1 in the blood began to increase slowly, and at 10 weeks, it increased by 30% compared with the control group. The content of bile acids in the bile began to increase significantly after 6 weeks of drug administration, and at 10 weeks, it increased by 28% compared with the control group.

[0055] Peptide sequence B group: After 3 weeks of drug administration, the ability of hepatocytes to uptake bilirubin began to increase, the content of bilirubin in the blood gradually decreased, and at 10 weeks, it decreased by 35% compared with the control group. The content of bilirubin in the bile increased significantly after 6 weeks of drug administration, and at 10 weeks, it increased by 32% compared with the control group. Peptide sequence C group: After 3 weeks of drug administration, the gallbladder contraction frequency began to increase, at 6 weeks, it increased by 22% compared with the control group, and at 10 weeks, it increased by 28%. The bile excretion volume increased significantly after 6 weeks of drug administration, and at 10 weeks, it increased by 32% compared with the control group.

[0056] Peptide sequence D group: After 3 weeks of drug administration, the oxidation and decomposition rate of bilirubin in the bile began to decrease, and at 6 weeks, it decreased by 30% compared with the control group.

[0057] Small molecule peptide composition group: The activity of CYP7A1 in the blood increased rapidly 3 weeks after administration, and was 45% higher than that of the control group at 10 weeks. The ability of hepatocytes to uptake bilirubin was significantly enhanced, and the bilirubin content in the blood decreased by 48% compared with the control group at 10 weeks. The gallbladder contraction frequency increased by 38% compared with the control group at 10 weeks, the bile excretion increased by 42%, and the bilirubin oxidation and decomposition rate decreased by 45%.

[0058] Control group: Calculi were formed in only 2 cows, with an average weight of 2 g.

[0059] Carrier control group: Calculi were formed in 3 cows, with an average weight of 2.3 g.

[0060] Peptide sequence A group: Calculi were formed in 5 cows, with an average weight of 4.2 g and a maximum weight of 5.8 g.

[0061] Peptide sequence B group: Calculi were formed in 6 cows, with an average weight of 4.4 g and a maximum weight of 6.2 g.

[0062] Peptide sequence C group: Calculi were formed in 5 cows, with an average weight of 4.3 g and a maximum weight of 6 g. Peptide sequence D group: Calculi were formed in 6 cows, with an average weight of 4.5 g and a maximum weight of 6.5 g.

[0063] Small molecule peptide composition group: Calculi were formed in 8 cows, with an average weight of 6 g and a maximum weight of 9 g.

[0064] Control group: There was obvious infiltration of inflammatory cells in the gallbladder tissue, and damage and erosion occurred in the gallbladder mucosa.

[0065] Carrier control group: The inflammation of the gallbladder tissue was mild, and a small amount of fibrous tissue encapsulation was formed around the microsphere carrier, which did not have a significant impact on the normal function of the gallbladder.

[0066] Peptide sequence A group, B group, C group and D group: The inflammation of the gallbladder tissue was extremely mild, the gallbladder mucosa was basically intact, and the microsphere carrier had good compatibility with the tissue.

[0067] Small molecule peptide composition group: There was almost no inflammatory manifestation in the gallbladder tissue, the gallbladder mucosa was smooth and structurally intact, and the tissue reaction around the microsphere carrier was weak.

[0068] In summary, the four small molecule peptide sequences extracted from Echinacea purpurea and the compositions composed of them can significantly promote the production of natural bezoar, effectively improving the yield and quality of bezoar. Among them, the effect of the small molecule peptide composition is the most significant. It can not only continuously release small molecule peptides through the implanted carrier, but also, under the synergistic effect of auxiliary intravenous injection, more effectively reduce gallbladder inflammation, creating a good internal environment for the formation of bezoar. At the same time, the composition can promote bile acid synthesis, bilirubin uptake and transport, gallbladder contraction and other physiological processes related to the formation of bezoar in multiple dimensions, thus greatly increasing the formation rate and quality of bezoar. This invention provides a new, highly efficient, safe and cost-controllable technical solution for solving the problem of short supply of natural bezoar, and has broad application prospects and great economic value in the field of biomedicine, especially in the traditional Chinese medicine industry.

Claims

1. A echinacea small molecule peptide composition for promoting the increased production of natural bezoar, characterized in that, Comprising the following small molecule peptide sequences: Peptide sequence A: Ala - Asp - Lys - Thr - Gly - Ser - Pro - Tyr - Leu - Ile - Val - Phe - Met - His; Peptide sequence B: Gly - Gln - Asn - Arg - Pro - Thr - Ser - Ile - Leu - Phe - Tyr - Trp - Lys - Glu; Peptide sequence C: Thr - Lys - Glu - Asp - Pro - Gly - Ser - Val - Ile - Leu - Phe - Tyr - Trp - His - Ala - Arg; Peptide sequence D: Pro - Arg - Glu - Asp - Gly - Ser - Thr - Ile - Val - Leu - Phe - Tyr - Trp - Asn - Gln - Lys; The said small molecule peptide sequences are derived from the extract of Echinacea.

2. The small molecule peptide composition according to claim 1, characterized in that, The content ratio of each peptide sequence is: peptide sequence A accounts for 15% - 35%, peptide sequence B accounts for 15% - 35%, peptide sequence C accounts for 15% - 35%, and peptide sequence D accounts for 15% - 35%.

3. A method for preparing a small molecule peptide composition according to any one of claims 1 - 2, characterized in that, Including the following steps: Collect the above - ground part of Echinacea with strong growth and no pests and diseases, wash it and cut it into small sections of 1 - 3 cm; Adopt vacuum freeze - drying technology to dry the small sections of Echinacea at - 35°C to - 25°C until the water content is below 7%, and crush it into a coarse powder that can pass through a 70 - 90 - mesh sieve; Use acetone, butanol, chloroform organic solvents and mixed solvents with different ratios, extract the coarse powder at an extraction temperature of 30°C - 50°C, time of 12 - 28 hours, and solid - liquid ratio of 1:5 (g / mL) - 1:9 (g / mL), collect the extract by centrifugation or filtration, and concentrate it under reduced pressure to obtain the concentrated extract; Preliminarily separate the concentrated extract of the extract through a macroporous adsorption resin column, select a suitable macroporous adsorption resin according to the polarity and molecular size of the peptide components, elute it with acetone solutions of different concentrations in a gradient manner, and collect the eluate that may contain peptide components; Further separate the above eluate by gel filtration chromatography, select Sephadex G - 50 and Sephacryl S - 100 gel media, separate according to the difference in peptide molecular size, and collect the eluate at different time periods for preliminary grouping; Re - separate the grouped peptide components by ion - exchange chromatography, select cation - exchange resin CM - Sepharose or anion - exchange resin DEAE - Sepharose according to the charge properties and quantity of peptide molecules, adjust the pH value and ionic strength of the eluate, and collect the solutions of different elution peaks to obtain relatively purified peptide components; Sequencing of relatively purified peptide components is performed using the Edman degradation method or mass spectrometry sequencing method, and peptide sequences A, B, C, and D are determined through bioinformatics analysis and mixed to obtain a small molecule peptide composition.

4. The preparation method according to claim 3, characterized in that, In the preliminary separation step of the macroporous adsorption resin column, polar macroporous adsorption resin is selected for peptide components with relatively high polarity, and non-polar or weakly polar macroporous adsorption resin is selected for non-polar or weakly polar peptide components.

5. The preparation method according to claim 3, characterized in that, In the mass spectrometry sequencing method, peptide samples are first pretreated by desalting, concentration, etc., and after injection into the mass spectrometer, ionization methods such as electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALDI) are selected, and professional software is used to analyze the mass spectrometry data.

6. Use of the small molecule peptide composition according to any one of claims 1 - 2 in promoting the production of natural bezoar.

7. The application according to claim 6, wherein The small molecule peptide composition is formulated into a solution and administered to cattle by intravenous injection at a dose of 3 mg / kg body weight, twice a week for 10 consecutive weeks.

8. A method for enhancing the production of natural bezoar, characterized in that Sodium alginate with good biocompatibility and biodegradability is selected to prepare microsphere carriers with a diameter of 1 - 2 mm. The small molecule peptide sequences described in claim 1 are respectively or mixed and loaded onto the microsphere carriers, and the microsphere carriers loaded with the small molecule peptide sequences or compositions are implanted near the gallbladder of cattle.

9. The synergistic method according to claim 8, characterized in that, The loading of small molecule peptide sequences onto microsphere carriers adopts physical adsorption or chemical coupling methods. In the physical adsorption method, the microsphere carriers and the small molecule peptide solution are mixed for a certain time under appropriate temperature and stirring conditions. In the chemical coupling method, covalent connection is achieved through chemical reactions between the surface active groups of the carrier and the specific functional groups of the small molecule peptide.

10. A pharmaceutical preparation, characterized in that, Comprising the small molecule peptide composition according to any one of claims 1 - 2 and a pharmaceutically acceptable carrier, excipient or diluent, and the dosage form is injection, capsule, tablet or granule.