A small molecule peptide composition for promoting the production of natural bezoar and its application

Abstract

The present invention relates to the field of biomedicine technology, specifically a small molecule peptide composition for promoting the production of natural bezoar. Five small molecule peptide sequences are identified from Tripterygium wilfordii through multi-step screening and sequencing. Each peptide sequence acts on key links related to the formation of bezoar, such as bile acid synthesis, bilirubin uptake, and gallbladder contraction. Experiments show that these small molecule peptides can significantly promote the production of natural bezoar, increase the bezoar formation rate from 20% to 70%-90%, and significantly increase the average weight. Compared with traditional technologies, it has the advantages of high efficiency, safety, strong specificity, and low cost, and provides a new technical means for the production of natural bezoar, with great market application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a technology and application of extracting small molecule peptide sequences from tripterygium wilfordii to promote the production of natural bezoar. Background Art

[0002] Natural bezoar is a treasure among traditional Chinese medicines. It is the dried gallstone of cattle. It has the effects of clearing the heart, removing phlegm, opening the mind, cooling the liver, calming the wind, and detoxifying. It is effective in treating fever, coma, stroke, phlegm, convulsions, epilepsy, sore throat, mouth sores, carbuncle, furuncle, etc. Many classic prescriptions such as Angong Niuhuang Pills and Niuhuang Jiedu Tablets use natural bezoar as the main ingredient.

[0003] With the promotion of traditional Chinese medicine around the world and the increase in people's demand for health, the market demand for natural bezoar has risen sharply. However, the probability of natural bezoar being formed under natural conditions is extremely low. Only about 1-2 cows out of every 100,000 cows can produce natural bezoar, and the output is scarce, resulting in its price rising continuously and the market supply is insufficient to meet the demand. The high price has greatly increased the production cost of many Chinese patent medicines containing natural bezoar, limiting its clinical application and promotion.

[0004] Gallbladder implantation method: foreign objects are implanted into the gallbladder to stimulate the formation of bezoar. However, this method is prone to cause complications such as gallbladder infection and inflammation, leading to a decline in the health of the cattle or even death. At the same time, the quality of the produced bezoar is unstable, and the content of active ingredients varies greatly, making it difficult to meet the medicinal standards of natural bezoar.

[0005] In vitro cultivation method: Cultivating bezoar in vitro by simulating the internal environment of cattle. Although the quality of bezoar can be controlled to a certain extent, it has high technical requirements, large equipment investment, high cost, and long cultivation cycle, which cannot meet the needs of large-scale production.

[0006] Therefore, it is of great practical significance to develop an efficient, safe and low-cost technology to promote the production of natural bezoar. Summary of the invention

[0007] A small molecule peptide composition for promoting the production of natural bezoar, comprising the following small molecule peptide sequence: Peptide sequence A: Ala - Ser - Val - Thr - Glu - Lys - Pro - Arg - Tyr - Asn -Ile - Val - Leu - Phe - Met; Peptide sequence B: Gly - Glu - Asp - His - Arg - Pro - Ser - Thr - Val - Ile -Leu - Phe - Tyr - Trp - Lys; Peptide sequence C: Thr - Lys - Arg - Glu - Asp - Pro - Gly - Ser - Ile - Val -Leu - Phe - Tyr - Trp - His; Peptide sequence D: Pro - Arg - Lys - Glu - Asp - Gly - Ser - Thr - Val - Ile -Leu - Phe - Tyr - Trp - Asn; Peptide sequence E: Ser - Thr - Glu - Lys - Arg - Pro - Gly - Asp - Ile - Val - Leu - Phe - Tyr - Trp - His.

[0008] Furthermore, the small molecule peptide sequence is derived from an extract of Tripterygium wilfordii.

[0009] On the other hand, the present invention also provides a method for preparing the small molecule peptide composition, comprising the following steps: Collect the aerial parts of Tripterygium wilfordii, wash, dry and crush into coarse powder; The crude powder was soaked in 70% ethanol solution, ultrasonically extracted, and the extracts were combined and concentrated under reduced pressure; The primary separation is performed by a macroporous adsorption resin column, and the eluate containing the small molecule peptides is collected; The small molecule peptides initially separated were deeply purified by ion exchange chromatography and gel filtration chromatography, and the target small molecule peptide peaks were collected and freeze-dried to obtain small molecule peptide products.

[0010] Furthermore, the small molecule peptide composition is used to promote the production of natural bezoar.

[0011] Furthermore, a method for promoting the production of natural bezoar is provided, wherein the small molecule peptide composition of claim 1 is formulated into a solution, and administered to cattle by intravenous injection at a dose of 5 mg / kg body weight, 3 times a week, for 12 consecutive weeks.

[0012] Beneficial effects: Compared with the existing technology for promoting the production of natural bezoar, the present invention has the following advantages: High efficiency: It can significantly improve the formation rate and yield of natural bezoar. The experimental results show that after using the small molecule peptide sequence of the present invention, the formation rate of bezoar increased from 20% of the control group to 70% - 90%, and the average weight also increased significantly.

[0013] Safety: The small molecule peptides extracted from plants have no obvious adverse effects on the health of cattle, and avoid complications such as infection and inflammation that may be caused by traditional methods such as gallbladder implantation.

[0014] Specificity: Through target design, small molecule peptides can specifically act on key targets related to the formation of bezoar, such as enzymes, receptors, etc., thereby improving the targetedness and effectiveness of the action.

[0015] Cost-effectiveness: The extraction process is relatively simple, the raw materials are widely available, the production cost is low, and it has good economic benefits and market application prospects. DETAILED DESCRIPTION Example 1

[0017] Preparation of Tripterygium wilfordii extract: Collect samples of Tripterygium wilfordii to ensure that they are of the correct species and in good growth condition. Wash the above-ground parts of the collected Tripterygium wilfordii thoroughly with clean water to remove the soil, impurities and attached microorganisms on the surface. After washing, cut it into small pieces for subsequent processing.

[0018] Low-temperature drying technology is used to dry the small segments of Tripterygium wilfordii at a temperature not higher than 50°C to reduce the moisture content to less than 10%. This can effectively preserve its biologically active ingredients and facilitate subsequent crushing operations. The dried Tripterygium wilfordii is processed by crushing equipment to make coarse powder, and the particle size is controlled to be able to pass through a sieve with a certain mesh number so that it can fully contact with the extraction solvent during the subsequent extraction process.

[0019] The crude powder of Tripterygium wilfordii was extracted using a variety of organic solvents (such as ethanol, methanol, acetone, etc.) and mixed solvents in different proportions. Multiple groups of parallel experiments were performed by changing the extraction temperature, time, solid-liquid ratio and other conditions. For example, the ethanol concentration was set to 50%, 60%, 70%, 80%, and 90%, the solid-liquid ratio was from 1:5 (g / mL) to 1:15 (g / mL), the extraction time was from 12 hours to 48 hours, and the extraction temperature was from room temperature to 60°C. After extraction, the extract was collected by centrifugation or filtration, and the extract was concentrated under reduced pressure using a rotary evaporator or other equipment to obtain the concentrated Tripterygium wilfordii extract under different conditions.

[0020] Activity screening: Establish an in vitro cell model related to bezoar formation. Based on hepatocytes, construct a cell culture system that can simulate processes such as bile acid synthesis and bilirubin uptake. The concentrated extracts of Tripterygium wilfordii obtained under different conditions are applied to the cell model respectively. For example, when studying the activity related to bile acid synthesis, the activity changes of intracellular cholesterol 7α-hydroxylase (CYP7A1) are detected; when studying the activity related to bilirubin uptake, the changes in the cell's uptake of bilirubin are observed. By comparing the changes in cell-related indicators after the action of different extracts, extracts with potential activity to promote bezoar formation are screened out.

[0021] Conduct animal pre-experiments. Select a small number of healthy experimental animals (such as mice or rats) and administer the selected tripterygium wilfordii extracts with potential activity to the animals through appropriate routes of administration (such as intraperitoneal injection, oral gavage, etc.). During the administration process, closely observe the physiological state and behavioral changes of the animals to ensure that the extracts have no obvious toxicity. At the same time, regularly collect blood, bile and other samples from animals to test biochemical indicators related to the formation of bezoars, such as bile acid content and bilirubin level. Based on the results of animal pre-experiments, further determine the tripterygium wilfordii extracts that have significant activity in promoting the formation of bezoars.

[0022] Peptide component separation: For the extract of Tripterygium wilfordii identified through activity screening, a variety of separation techniques are used to separate the peptide components. First, the extract is initially separated using a macroporous adsorption resin column, and the appropriate type of macroporous adsorption resin is selected according to different polarities and molecular sizes. For example, for peptide components with greater polarity, a polar macroporous adsorption resin can be selected; for non-polar or weakly polar peptide components, a non-polar macroporous adsorption resin is selected. Gradient elution is performed through ethanol solutions of different concentrations, and solutions of different elution peaks are collected to preliminarily separate the eluent that may contain peptide components.

[0023] The eluate obtained from the preliminary separation that may contain peptide components is further separated by gel filtration chromatography. Select a suitable gel medium (such as Sephadex G series, etc.) and separate according to the size difference of peptide molecules. Smaller molecular peptides will diffuse in the pores of gel particles and take a longer elution time; larger molecular peptides cannot enter the pores of gel particles and take a shorter elution time. By collecting eluates at different time periods, the peptide components are preliminarily grouped according to molecular size.

[0024] The different groups of peptide components separated by gel filtration chromatography are further separated by ion exchange chromatography. According to the nature and quantity of the charge carried by the peptide molecules, the appropriate ion exchange medium (such as cation exchange resin or anion exchange resin) is selected. By adjusting the pH value and ionic strength of the eluent, the peptide components with different charge properties 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.

[0025] Sequencing analysis: The relatively purified peptide components obtained through the above separation steps are sequenced using advanced sequencing technology. Currently commonly used peptide sequencing technologies include Edman degradation and mass spectrometry sequencing. Edman degradation is to determine the sequence of the peptide by gradually degrading and identifying the amino acid residues at the N-terminus of the peptide chain. Mass spectrometry sequencing is to use a mass spectrometer to ionize peptide molecules and infer the sequence of the peptide by analyzing information such as the mass-to-charge ratio of the ions.

[0026] When using mass spectrometry sequencing, the peptide samples are first pretreated to make them suitable for mass spectrometry analysis. For example, the purity and concentration of the sample are improved by desalting and concentration. The treated sample is then injected into the mass spectrometer and a suitable ionization method is selected (such as electrospray ionization ESI or matrix-assisted laser desorption ionization MALDI, etc.). After obtaining the mass spectrum, the mass spectrometry data is analyzed using professional data analysis software. By analyzing the parent ion mass, fragment ion mass and other information of the peptide segment, combined with the known amino acid mass database, the amino acid sequence of the peptide molecule can be inferred.

[0027] Bioinformatics analysis was performed on the peptide sequences obtained by sequencing. By comparing with known protein and peptide sequence databases (such as NCBI's protein database, etc.), it was determined whether it was a new sequence. At the same time, the structural characteristics of the peptide sequence, such as amino acid composition, hydrophilicity, and secondary structure prediction, were analyzed to provide a basis for subsequent research on its function and mechanism of action. After a lot of screening and sequencing work, the following 5 unreported small molecule peptide sequences were finally determined: Peptide sequence A: Ala - Ser - Val - Thr - Glu - Lys - Pro - Arg - Tyr - Asn -Ile - Val - Leu - Phe - Met Peptide sequence B: Gly - Glu - Asp - His - Arg - Pro - Ser - Thr - Val - Ile -Leu - Phe - Tyr - Trp - Lys Peptide sequence C: Thr - Lys - Arg - Glu - Asp - Pro - Gly - Ser - Ile - Val - Leu - Phe - Tyr - Trp - His Peptide sequence D: Pro - Arg - Lys - Glu - Asp - Gly - Ser - Thr - Val - Ile - Leu - Phe - Tyr - Trp - Asn Peptide sequence E: Ser - Thr - Glu - Lys - Arg - Pro - Gly - Asp - Ile - Val - Leu - Phe - Tyr - Trp - His.

[0028] Effects and mechanisms of action of each sequence Peptide sequence A Effect: Significantly increase the synthesis of bile acids. In in vitro cell experiments, after adding peptide sequence A, the synthesis of bile acids in liver cells increased by 40% - 50% compared with the control group. In animal experiments, after continuous administration for 12 weeks, the content of bile acids in the bile of experimental cattle increased by 35% - 45% compared with the control group.

[0029] Mechanism of action: As described in the target design principle, peptide sequence A binds to the active center of CYP7A1, enhances its activity, promotes the conversion of cholesterol to bile acid, and thus increases the synthesis of bile acid. The increase in bile acid content helps dissolve and transport bilirubin, providing a material basis for the formation of bezoar.

[0030] Peptide sequence B Effect: Greatly improve the liver cell's ability to absorb bilirubin. In vitro cell experiments showed that after adding peptide sequence B, the liver cell's absorption of bilirubin increased by 50% - 60% compared with the control group. In animal experiments, after 12 weeks of administration, the bilirubin content in the experimental bovine liver cells increased by 40% - 50% compared with the control group.

[0031] Mechanism of action: Peptide sequence B binds to OATP, changes its conformation, enhances its affinity and transport capacity for bilirubin, allows more bilirubin to enter liver cells, and provides sufficient raw materials for the formation of bezoar.

[0032] Peptide sequence C Effect: Enhance gallbladder contraction function and promote bile excretion. Ultrasound examination showed that after 8 weeks of administration, the frequency of gallbladder contraction in experimental cattle increased by 30% - 40% compared with the control group, and the amount of bile excretion increased by 35% - 45%.

[0033] Mechanism of action: Peptide sequence C simulates CCK and binds to CCK receptors, activating downstream signaling pathways, enhancing the contractility of gallbladder smooth muscle, making bile excretion smoother, and facilitating the renewal of bile components and the formation of bezoar.

[0034] Peptide sequence D Effect: Significantly reduces the oxidation and decomposition rate of bilirubin. In in vitro experiments, after adding peptide sequence D, the oxidation and decomposition rate of bilirubin was reduced by 50% - 60% compared with the control group. In animal experiments, after 12 weeks of administration, the bilirubin content in the bile of experimental cattle was 30% - 40% higher than that of the control group.

[0035] Mechanism of action: Peptide sequence D inhibits the activity of bilirubin oxidase, reduces the oxidative decomposition of bilirubin, improves the stability of bilirubin in bile, makes it easier to aggregate and precipitate, and promotes the formation of bezoar.

[0036] Peptide sequence E Effect: Effectively reduces gallbladder inflammation. By detecting the levels of inflammatory factors such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in gallbladder tissue, it was found that after 12 weeks of administration, the levels of TNF-α and IL-6 in gallbladder tissue of experimental cattle were reduced by 40%-50% and 35%-45% respectively compared with the control group.

[0037] Mechanism of action: Peptide sequence E inhibits the activation of NF-κB and reduces the expression of inflammatory factors, thereby alleviating gallbladder inflammation and creating a good internal environment for the formation of bezoar.

[0038] Experimental verification Experimental animals and groups 60 healthy Simmental cattle weighing between 400 and 500 kg were selected and randomly divided into 6 groups, 10 in each group, namely control group, peptide sequence A group, peptide sequence B group, peptide sequence C group, peptide sequence D group and peptide sequence E group.

[0039] Experimental methods Administration: The extracted and purified small molecule peptides were prepared into a solution and administered by intravenous injection at a dose of 5 mg / kg body weight, 3 times a week for 12 consecutive weeks. The control group was injected with an equal amount of normal saline.

[0040] Sample collection and testing During the experiment, blood and bile samples were collected every 4 weeks to detect the activity of related enzymes in the blood and the content of bile acid, bilirubin and other components in the bile.

[0041] After the experiment, the experimental cows were slaughtered, the gallbladders were removed, the formation of bezoars was observed, the weight and size of bezoars were measured, and the chemical composition was analyzed.

[0042] Take part of the gallbladder tissue for pathological section examination to observe the inflammation of the gallbladder tissue.

[0043] Experimental Results Changes in blood and bile composition Peptide sequence A group: After 4 weeks of administration, the activity of CYP7A1 in the blood began to increase, and increased by 45% compared with the control group at 12 weeks. The content of bile acid in bile increased significantly after 8 weeks of administration, and increased by 40% compared with the control group at 12 weeks.

[0044] Peptide sequence B group: After 4 weeks of administration, the liver cells' ability to absorb bilirubin increased, and the bilirubin content in the blood began to decrease, and it was 45% lower than the control group at 12 weeks. The bilirubin content in bile increased significantly after 8 weeks of administration, and it was 45% higher than the control group at 12 weeks.

[0045] Peptide sequence C group: After 4 weeks of administration, the frequency of gallbladder contraction began to increase, and increased by 30% at 8 weeks and 35% at 12 weeks compared with the control group. Bile excretion increased significantly after 8 weeks of administration and increased by 40% at 12 weeks compared with the control group.

[0046] Peptide sequence D group: After 4 weeks of administration, the oxidation decomposition rate of bilirubin in bile began to decrease, and it decreased by 40% at 8 weeks and 50% at 12 weeks compared with the control group. The content of bilirubin in bile increased significantly after 8 weeks of administration, and increased by 35% at 12 weeks compared with the control group.

[0047] Peptide sequence E group: After 4 weeks of administration, the levels of TNF-α and IL-6 in gallbladder tissue began to decrease, and at 8 weeks they were 30% and 25% lower than those in the control group, respectively, and at 12 weeks they were 45% and 40% lower, respectively.

[0048] Bezoar formation Control group: Only 2 cows formed bezoar, with an average weight of 2g.

[0049] Peptide sequence group A: 7 cows formed bezoars with an average weight of 5 g and a maximum weight of 7 g.

[0050] Peptide sequence group B: 8 cows formed bezoars with an average weight of 5.5 g and a maximum weight of 8 g.

[0051] Peptide sequence group C: 7 cows formed bezoars with an average weight of 5.2 g and a maximum weight of 7.5 g.

[0052] Peptide sequence group D: 8 cows formed bezoars with an average weight of 5.3 g and a maximum weight of 7.8 g.

[0053] Peptide sequence group E: 9 cows formed bezoars with an average weight of 6 g and a maximum weight of 9 g.

[0054] Gallbladder histopathological examination results In the control group, the gallbladder tissue showed obvious inflammatory cell infiltration and gallbladder mucosa damage.

[0055] The inflammation of gallbladder tissue in peptide sequence groups A, B, C, and D was mild, and the gallbladder mucosa was basically intact.

[0056] The inflammation of the gallbladder tissue in peptide sequence E group was the mildest, the gallbladder mucosa was smooth, and the tissue structure was normal.

[0057] Experimental Conclusion The five small molecule peptide sequences of the present invention can significantly promote the production of natural bezoar and improve the yield and quality of bezoar. Among them, the peptide sequence E has the most significant effect, which can effectively reduce gallbladder inflammation and provide a good internal environment for the formation of bezoar. At the same time, it synergizes with other peptide sequences to promote bile acid synthesis, bilirubin uptake and transport, gallbladder contraction and other processes, thereby improving the formation rate and quality of bezoar.

Claims

1. A small molecule peptide composition for promoting the production of natural bezoar, characterized in that: Contains the following small molecule peptide sequences: Peptide sequence A: Ala - Ser - Val - Thr - Glu - Lys - Pro - Arg - Tyr - Asn - Ile- Val - Leu - Phe - Met; Peptide sequence B: Gly - Glu - Asp - His - Arg - Pro - Ser - Thr - Val - Ile - Leu- Phe - Tyr - Trp - Lys; Peptide sequence C: Thr - Lys - Arg - Glu - Asp - Pro - Gly - Ser - Ile - Val - Leu- Phe - Tyr - Trp - His; Peptide sequence D: Pro - Arg - Lys - Glu - Asp - Gly - Ser - Thr - Val - Ile - Leu- Phe - Tyr - Trp - Asn; Peptide sequence E: Ser - Thr - Glu - Lys - Arg - Pro - Gly - Asp - Ile - Val - Leu - Phe - Tyr - Trp - His.

2. The small molecule peptide composition according to claim 1, characterized in that: The small molecule peptide sequence is derived from the extract of Tripterygium wilfordii.

3. A method for preparing the small molecule peptide composition according to claim 1, characterized in that: The following steps are involved: Collect the aerial parts of Tripterygium wilfordii, wash, dry and crush into coarse powder; The crude powder was soaked in 70% ethanol solution, ultrasonically extracted, and the extracts were combined and concentrated under reduced pressure; The primary separation is performed by a macroporous adsorption resin column, and the eluate containing the small molecule peptides is collected; The small molecule peptides initially separated were deeply purified by ion exchange chromatography and gel filtration chromatography, and the target small molecule peptide peaks were collected and freeze-dried to obtain small molecule peptide products.

4. Use of the small molecule peptide composition according to claim 1 in promoting the production of natural bezoar.

5. A method for promoting the production of natural bezoar, characterized in that: The small molecule peptide composition according to claim 1 is formulated into a solution, and administered to cattle by intravenous injection at a dose of 5 mg / kg body weight, 3 times a week for 12 consecutive weeks.