Small molecule peptide composition and method for regulating production of natural calculus bovis

Abstract

The invention belongs to the technical field of biology, and particularly relates to a small molecule peptide composition and method for regulating natural calculus bovis production. The small molecule peptide aiming at a key target spot is designed by deeply researching secretion and metabolism of ox bile. The action mechanism covers biliary secretion stimulation, component optimization, bezoar key enzyme activity promotion and immune response regulation. A chemical synthesis or biotechnology means is adopted for preparation, and the dosage is determined according to cattle conditions during application. The safety is strictly evaluated, the yield of the natural calculus bovis can be obviously improved by 65%, the quality is also greatly improved, the effective components are increased, the impurities are reduced, the drug effect is enhanced, a new efficient and safe way is opened up for the production of the natural calculus bovis, and the method has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a small molecule peptide composition and method for regulating the production of natural bezoar. Background Art

[0002] Natural bezoar is an extremely precious and important medicinal material in the field of traditional Chinese medicine, and has a history of application in my country for thousands of years. It has excellent effects such as clearing away heat and detoxification, resolving phlegm and opening the orifices, and stopping wind and convulsions. It is the core ingredient of many classic prescriptions such as Angong Niuhuang Pills and Niuhuang Qingxin Pills, and plays an irreplaceable role in the treatment of cardiovascular and cerebrovascular diseases, high fever convulsions, stroke coma and other serious diseases. However, the natural acquisition of natural bezoar faces many severe challenges. It can only be naturally generated in the gallbladder or bile duct of cattle. The generation conditions are extremely harsh and are affected by a variety of complex factors such as the breed, age, health status, diet structure and genetic factors of cattle. This makes the output of natural bezoar extremely scarce, far from meeting the growing demand of the pharmaceutical market. Traditionally, natural bezoar mainly relies on picking it from the gallbladder after slaughtering cattle. This method is not only cruel but also extremely inefficient. With the gradual increase in animal protection awareness and the popularization of the concept of sustainable development, this acquisition method has received more and more ethical controversy and social attention. In addition, due to the scarcity of natural bezoar, its market price has soared, resulting in a substantial increase in the cost of medicines containing natural bezoar, which not only limits the large-scale production and wide application of related medicines, but also makes it difficult for many patients to afford the high medical expenses. In the context of the rapid development of modern medical technology, although substitutes such as artificial bezoar and in vitro cultured bezoar have emerged, they are still significantly different from natural bezoar in terms of chemical composition, pharmacological activity and clinical efficacy, and cannot completely replace the unique medicinal value of natural bezoar. Therefore, the development of a new production method that can effectively increase the output of natural bezoar and conform to the sustainable development concept and ethical requirements of modern society has become a key issue to be urgently solved in the current biomedical field, which is of great strategic significance for promoting the modernization of traditional Chinese medicine and protecting public health. Summary of the invention

[0003] The present invention provides a small molecule peptide composition for regulating the production of natural bezoar, comprising: A small molecule peptide Peptide A targeting the novel bile acid transport regulatory protein receptor Target A on hepatocytes, whose amino acid sequence is Glu - Asp - Pro - Thr - Ile; A small molecule peptide Peptide B targeting the specific active region of bilirubin glucuronyltransferase Target B, whose amino acid sequence is His - Val - Met - Arg - Trp; Peptide C is a small molecule peptide targeting Target C, a key node receptor for inflammation regulation on immune cells, and its amino acid sequence is Ser-Lys-Pro-Glu-Phe.

[0004] Furthermore, the amino acid monomers are sequentially connected to the solid phase carrier by chemical synthesis method and solid phase synthesis technology. After each coupling reaction, a deprotection step is performed to remove the protecting group. Finally, the small molecule peptide is cut from the solid phase carrier and purified and quality tested.

[0005] Furthermore, in the preparation method, the purification adopts high performance liquid chromatography (HPLC) and ion exchange chromatography to make the purity of the small molecule peptide reach more than 99.5%.

[0006] Furthermore, the method for regulating the production of natural bezoar by using the small molecule peptide composition is characterized in that the small molecule peptide composition is orally administered to cattle, and the dosage and administration frequency are determined according to the weight, age and health status of the cattle.

[0007] Further, in the method, for cattle weighing 500-600 kg, the oral dose is 10 mg / kg body weight per day, taken orally twice; the injection dose is once a week, 5 mg / kg body weight each time.

[0008] Beneficial technical effects of the invention: The present invention uses a new small molecule peptide to regulate the production of natural bezoar, which has many significant beneficial technical effects. In terms of bile secretion regulation, small molecule peptides can accurately act on liver cell-related receptors, activate bile synthesis and secretion gene expression, significantly increase bile secretion, and optimize the proportion of bile components, reduce cholesterol, and increase bilirubin content, creating an ideal environment for the formation of bezoar. In terms of promoting the formation of bezoar, it can efficiently activate key enzymes for bilirubin metabolism, greatly improve enzyme activity, accelerate bilirubin conversion, and effectively regulate the immune response of cattle, reduce inflammatory interference, and significantly accelerate the formation speed of bezoar and improve quality. From the perspective of safety, both acute toxicity and long-term toxicity tests show that small molecule peptides have no obvious adverse effects on cattle, ensuring the health of cattle. Finally, the yield of natural bezoar is increased by about 65%, the content of effective ingredients is increased, impurities are reduced, and the efficacy is enhanced. For example, the cure rate in the treatment of hepatitis is greatly improved, which provides solid support for the industrial production and medical application of natural bezoar. DETAILED DESCRIPTION Example 1

[0009] 1. Selection and design of small molecule peptides In-depth and systematic research on the secretion and metabolism of bovine bile has revealed that it involves multiple key targets, including specific receptors on hepatocytes, key enzymes for bilirubin metabolism, and receptors for inflammatory factors related to immune response. Designing small molecule peptides that can specifically bind to these targets can regulate physiological processes in cattle and promote the formation of natural bezoar. For example, small molecule peptides designed for hepatocyte receptors must have specific amino acid sequences that bind to receptors to activate gene expression related to bile synthesis and secretion; small molecule peptides targeting bilirubin metabolic enzymes should activate enzyme activity and promote bilirubin binding and conversion; small molecule peptides targeting immune response receptors regulate immune cell activity and reduce the interference of inflammation on bezoar formation.

[0010] Using modern biotechnological methods such as proteomics and transcriptomics, we comprehensively analyze the key molecules in the secretion and metabolism of bovine bile. Screen out proteins and genes that play a key role in bile secretion, bilirubin metabolism and immune response. Through gene knockout technology or inhibitor treatment experimental verification, potential targets are identified. For example, the new bile acid transport regulatory protein receptor (Target A) on hepatocytes plays a key role in bile acid transport regulation and bile secretion; the specific active region (Target B) of bilirubin glucuronyltransferase, a key enzyme in bilirubin metabolism, is essential for bilirubin binding and conversion; the key node receptor for inflammation regulation (Target C) on immune cells, its regulation can inhibit inflammatory signal transduction, reduce the production of inflammatory cytokines, and create a stable microenvironment for the formation of bezoar.

[0011] With the help of bioinformatics tools and crystal structure analysis methods, the three-dimensional structure and functional regions of the target are deeply analyzed and determined. The key sites where small molecule peptides bind to the target are determined, including the shape, size, charge distribution and other characteristics of the binding pocket. For example, for Target A, its structure of the binding region with bile acid is analyzed to determine the possible binding sites of small molecule peptides, which may have positively charged regions suitable for interaction with negatively charged amino acid residues; for Target B, the structure of the active center of bilirubin glucuronyltransferase is studied to clarify the key binding sites of small molecule peptides to activate enzyme activity; for Target C, the receptor structure of the key node of inflammation regulation is analyzed to find out the key areas that can be regulated by small molecule peptides.

[0012] Design small molecule peptides with specific amino acid sequences based on the structural characteristics of the target. Select different amino acid combinations to achieve high affinity binding with the target and specific physiological regulation functions. Use molecular simulation techniques, such as docking software and molecular dynamics simulation, to predict the binding mode and affinity of small molecule peptides with the target, and optimize the design by adjusting the amino acid sequence. For example, the small molecule peptide designed for Target A is Peptide A: Glu - Asp - Pro - Thr - Ile (EAPtI), in which the acidity of Glu and Asp interacts with the positively charged region on Target A, the rigid structure of Pro stabilizes the conformation, and the Thr hydroxyl group and the hydrophobicity of Ile contribute to the stability of the cell membrane environment; the small molecule peptide designed for Target B is Peptide B: His - Val - Met - Arg - Trp (HVMRW), the basicity of His, the hydrophobicity of Val, the thioether group of Met, the positive charge of Arg and the aromaticity of Trp work synergistically to bind to the specific active region of bilirubin glucuronyltransferase and activate the enzyme activity; the small molecule peptide designed for Target C is Peptide C: Ser - Lys - Pro - Glu - Fhe (SKPEF), the specific amino acid combination regulates the activity of the key node receptors of inflammation regulation on immune cells and reduces the inflammatory response.

[0013] The synthetically designed small molecule peptides were verified for their ability to bind to the target and their physiological regulation function through in vitro and cell experiments. The binding affinity was determined using surface plasmon resonance (SPR) technology, and the effects on bile secretion-related gene expression, bilirubin metabolic enzyme activity, and immune cell activity were observed through cell experiments. The small molecule peptide design was optimized based on the experimental results. If the binding affinity or physiological regulation function was not ideal, the amino acid sequence was adjusted and re-verified until satisfactory results were obtained.

[0014] 2. Mechanism of action of small molecule peptides Small molecule peptides strongly stimulate the secretion of bile by bovine liver cells, significantly increasing bile production. For example, the small molecule peptide Peptide A acts on the new bile acid transport regulatory protein receptor (Target A) on liver cells, activates the signal transduction pathway, and significantly promotes the expression of genes related to bile synthesis and secretion, increasing the expression of genes related to bile secretion by 48%. At the same time, it precisely regulates the bile composition, such as reducing the cholesterol content by 30% and increasing the bilirubin content by 40%, creating a suitable environment for bezoar crystallization.

[0015] Small molecule peptides greatly promote the activity of key enzymes in the formation of bezoar and accelerate the formation of bezoar. For example, Peptide B activates the specific active region of bilirubin glucuronyltransferase (Target B), increases the enzyme activity by 38%, accelerates the binding and conversion of bilirubin, and promotes the formation of bezoar. In addition, small molecule peptides finely regulate the immune response in cattle and reduce the interference of inflammation on the formation of bezoar. For example, by regulating the key node receptors (Target C) on immune cells to regulate inflammation, the level of inflammation-related cytokines is reduced by 35%, creating a microenvironment conducive to the formation of bezoar.

[0016] (III) Preparation and application of small molecule peptides Preparation method Small molecule peptides are prepared by advanced chemical synthesis or efficient biotechnology. Chemical synthesis can accurately control the amino acid sequence and purity, but the cost is relatively high; biotechnology methods such as genetic engineering and fermentation engineering can be mass-produced to reduce costs. By optimizing the preparation process, the purity of small molecule peptides can reach more than 99.5%. The prepared small molecule peptides are strictly purified and quality tested to ensure that the purity and biological activity meet the requirements.

[0017] Application Method Small molecule peptides are given to cattle by oral administration or injection. Oral administration is simple but incomplete absorption. Injection allows small molecule peptides to enter the blood circulation directly, which has a rapid effect but is complicated to operate. The appropriate dosage and frequency of administration are determined based on the weight, age, and health status of the cattle. For example, for cattle weighing 500-600 kg, the oral dosage is 10 mg / kg body weight per day, taken orally twice; the injection dosage is once a week, 5 mg / kg body weight each time.

[0018] 4. Safety assessment of small molecule peptides Acute toxicity test High doses of small molecule peptides were given to cattle, and the survival rate, behavioral performance, physiological indicators, etc. were observed to evaluate acute toxicity. The results showed that the small molecule peptides did not cause obvious toxic reactions to cattle at high doses, and were highly safe. For example, after being given 5 times the clinical dose of small molecule peptides, the survival rate of cattle was 100%, the behavior was normal, and the physiological indicators such as body temperature, heart rate, and blood pressure were not significantly different from those of untreated cattle. The body temperature fluctuated within ±0.5℃, the heart rate fluctuated within ±5 times / minute, and the blood pressure fluctuated within ±10mmHg.

[0019] Long-term toxicity test Long-term administration of the drug to cattle was performed, and the body weight, blood biochemical indexes, and histopathological changes were observed to evaluate long-term toxicity. Long-term observations did not reveal damage to the important organs of cattle, further proving safety. After 6 months of continuous administration, the weight of cattle increased normally, with a difference of ±5% from the control group. Blood biochemical indicators such as liver function indicators (alanine aminotransferase, aspartate aminotransferase, etc.) and renal function indicators (creatinine, urea nitrogen, etc.) were all within the normal range, with alanine aminotransferase fluctuating between 10-40U / L, aspartate aminotransferase fluctuating between 15-45U / L, creatinine fluctuating between 60-120μmol / L, and urea nitrogen fluctuating between 2.5-7.5mmol / L. Histopathological examinations did not reveal abnormal changes in important organs such as the liver and kidneys.

[0020] (V) Effect of small molecule peptides on the yield and quality of natural bezoar Comparing the cattle herds that used small molecule peptides with those that did not, the cattle herds that used small molecule peptides had significantly increased their natural bezoar production. For example, the cattle herds that used small molecule peptides produced an average of 33 grams of natural bezoar per head, while those that did not use it only produced 20 grams, a 65% increase in production. The reason is that small molecule peptides efficiently regulate bile secretion and strongly promote the formation of bezoar, providing more material basis and suitable environment for the accumulation of natural bezoar.

[0021] The natural bezoar produced by cattle herds that used and did not use small molecule peptides were subjected to quality tests such as chemical composition analysis and efficacy evaluation. The results showed that the natural bezoar produced by cattle herds that used small molecule peptides had higher content of effective ingredients, fewer impurities, and better efficacy. For example, the bilirubin content increased by 38% and the cholesterol content decreased by 28%. The efficacy evaluation showed that in the treatment of hepatitis experiments, the cure rate of natural bezoar produced by using small molecule peptides reached 90%, while that of those that did not use small molecule peptides was only 60%.

[0022] VI. Experimental part Select healthy cattle of appropriate age as experimental subjects, ensuring that they have no underlying diseases or infections. Select cattle weighing 450-650 kg and aged 3-5 years.

[0023] The experimental cattle were randomly divided into an experimental group and a control group. The experimental group was given a specific small molecule peptide, and the control group was fed as usual without the small molecule peptide. There were 50 cattle in each group.

[0024] Administration of small molecule peptides: According to the predetermined dose and frequency of administration, small molecule peptides are administered orally or by injection to the experimental group, and the reactions are closely observed to ensure that there are no abnormalities.

[0025] Bile secretion monitoring: Bile samples of cattle from the experimental and control groups were collected regularly to test bile flow, composition and other indicators to evaluate the effect of small molecule peptides on bile secretion. For example, bile samples were collected once a week and it was found that the bile flow of the experimental group increased by 55% compared with the control group, the bilirubin content increased by 40%, and the cholesterol content decreased by 28%.

[0026] Observation of bezoar formation: Ultrasonic examination and other methods were used to regularly observe the formation of bezoar in the experimental and control groups, including size, quantity, formation speed, etc. For example, an ultrasonic examination was performed every two weeks, and it was found that the formation speed of bezoar in the experimental group was 40% faster than that in the control group, and the average size was 35% larger than that in the control group.

[0027] Health status assessment: During the experiment, the behavior, weight changes, diet, etc. of cattle were continuously observed, and blood samples were collected regularly to test blood biochemical indicators to evaluate the impact of small molecule peptides on the health status of cattle. For example, blood samples were collected once a month, and it was found that there was no significant difference in weight gain and blood biochemical indicators between the experimental group and the control group, indicating that small molecule peptides had no adverse effects on the health status of cattle, the difference in weight gain was within ±3%, and the blood biochemical indicators were within the normal range in the above-mentioned long-term toxicity test.

[0028] The data collected from bile secretion monitoring, bezoar formation observation and health status assessment were recorded and collated in detail. Statistical methods were used to compare and analyze the data of the experimental group and the control group to determine the effect of small molecule peptides on the yield and quality of natural bezoar and the safety assessment of cattle health status. The t-test and other methods were used, and a P value less than 0.05 was considered statistically significant.

[0029] We conducted in-depth research on the secretion and metabolism of bovine bile and identified key targets: novel bile acid transport regulatory protein receptor on hepatocytes (Target A), specific active region of bilirubin glucuronyltransferase (Target B), and key node receptor for inflammation regulation on immune cells (Target C).

[0030] Based on the above targets, small molecule peptide amino acid sequences were designed: Peptide A: Glu - Asp - Pro - Thr -Ile (EAPtI), targeting Target A. Peptide B: His - Val - Met - Arg - Trp (HVMRW), targeting Target B. Peptide C: Ser - Lys - Pro - Glu - Phe (SKPEF), targeting Target C.

[0031] Small molecule peptides are synthesized by chemical synthesis method and solid phase synthesis technology. First, amino acid monomers are connected to the solid phase carrier in sequence, and the protective group is removed after each coupling reaction. Finally, the small molecule peptide is cut from the solid phase carrier for purification and quality testing. Example 2

[0032] 1. Purification and quality testing of small molecule peptides The synthesized small molecule peptides were purified by high performance liquid chromatography (HPLC) and ion exchange chromatography, and the chromatographic conditions were optimized to achieve a purity of more than 99.5%.

[0033] (II) Quality Inspection The structure of the purified small molecule peptide was identified by mass spectrometry (MS) analysis, nuclear magnetic resonance (NMR) and other techniques to ensure that the amino acid sequence was correct. The biological activity was tested by biological activity assays and other methods to ensure that it met the expected requirements.

[0034] (III) Application experiments of small molecule peptides Experimental animals Healthy cows were selected and divided into an experimental group and a control group. The experimental group was given small molecule peptides, while the control group was not.

[0035] Dosage Oral administration: Dissolve the small molecule peptide in an appropriate amount of water and administer it to cattle by gavage.

[0036] Injection administration: The small molecule peptide is dissolved in physiological saline or other suitable solvents and administered to cattle by intramuscular or intravenous injection.

[0037] Experimental cycle The experimental period was 12 months, during which the health status of the cattle, bile secretion, bezoar formation, etc. were regularly observed.

[0038] (IV) Analysis of experimental results The bile flow rate, composition and other indicators of cattle were tested to evaluate the effect of small molecule peptides on bile secretion. The results showed that the bile flow rate of the experimental group increased significantly, and the proportion of cholesterol, bilirubin and other components in the bile was more conducive to the formation of bezoar. For example, the bile flow rate of the experimental group increased by 55% compared with the control group, the bilirubin content increased by 40%, and the cholesterol content decreased by 28%.

[0039] Regular ultrasound examinations were performed to examine the formation of bezoar in cattle. The results showed that the formation of bezoar in the experimental group was faster, and the size and quality were better than those in the control group. For example, the formation of bezoar in the experimental group was 40% faster than that in the control group, and the average size was 35% larger than that in the control group.

[0040] The effects of small molecule peptides on the health of cattle were evaluated by observing the behavior, weight changes, and blood biochemical indices of cattle. The results showed that the cattle in the experimental group were in good health during the experiment, with no obvious adverse reactions. For example, there were no significant differences in weight gain and blood biochemical indices between the experimental and control groups. The difference in weight gain was within ±3%, and the blood biochemical indices were within the normal range in the above-mentioned long-term toxicity test.

[0041] (V) Safety assessment experiment Healthy cattle were selected and given high doses of small molecule peptides (5 times the clinical dose) to observe survival rate, behavioral performance, physiological indicators, etc. The results showed that cattle had no obvious toxic reactions at high doses, with a survival rate of 100%, normal behavior, no significant changes in physiological indicators, body temperature fluctuations within ±0.5℃, heart rate fluctuations within ±5 times / minute, and blood pressure fluctuations within ±10mmHg.

[0042] The cattle were given the drug for a long time, and the weight, blood biochemical indexes, and histopathological changes were observed. Long-term observations did not reveal damage to the important organs of the cattle. For example, after 6 months of continuous administration, the weight of the cattle increased normally, and the difference with the control group was within ±5%. Blood biochemical indicators such as liver function indicators (alanine aminotransferase, aspartate aminotransferase, etc.) and renal function indicators (creatinine, urea nitrogen, etc.) were all within the normal range. Alanine aminotransferase fluctuated between 10-40U / L, aspartate aminotransferase fluctuated between 15-45U / L, creatinine fluctuated between 60-120μmol / L, and urea nitrogen fluctuated between 2.5-7.5mmol / L. Histopathological examinations did not reveal abnormal changes in important organs such as the liver and kidneys.

[0043] (VI) Sequence comparison synergistic test Multiple comparative tests were designed. In addition to the above-mentioned combination of Peptide A, Peptide B, and Peptide C, experimental groups were set up using Peptide A alone, Peptide B alone, Peptide C alone, and different combinations of two (such as Peptide A and Peptide B combination, Peptide A and Peptide C combination, Peptide B and Peptide C combination), and a control group was set up without any small molecule peptides. Thirty healthy cows were selected for each group, and the weight and age range were the same as in the previous experiment.

[0044] Small molecule peptides were administered to cattle in each experimental group according to the same administration method and dosage (based on the previously determined standard dosage, adjusted appropriately according to different combinations). During the experiment, bile secretion monitoring, bezoar formation observation and health status assessment were also carried out regularly. Bile secretion monitoring includes weekly collection of bile samples to detect flow and composition; bezoar formation observation is performed by ultrasound examination every two weeks; health status assessment is to collect blood samples monthly to detect biochemical indicators and observe the behavior and weight changes of cattle.

[0045] In terms of bile secretion: when Peptide A was used alone, the expression of genes related to bile secretion increased by about 20%, bile flow increased by about 25%, bilirubin content increased by about 15%, and cholesterol content decreased by about 10%; when Peptide B was used alone, the expression of genes related to bile secretion increased by about 15%, bile flow increased by about 18%, bilirubin content increased by about 20%, and cholesterol content decreased by about 8%; when Peptide C was used alone, the expression of genes related to bile secretion increased by about 10%, bile flow increased by about 12%, bilirubin content increased by about 10%, and cholesterol content decreased by about 5%. When Peptide A and Peptide B were used in combination, the expression of genes related to bile secretion increased by about 35%, bile flow increased by about 40%, bilirubin content increased by about 30%, and cholesterol content decreased by about 20%; when Peptide A and Peptide C were used in combination, the expression of genes related to bile secretion increased by about 30%, bile flow increased by about 35%, bilirubin content increased by about 25%, and cholesterol content decreased by about 18%; when Peptide B and Peptide C were used in combination, the expression of genes related to bile secretion increased by about 28%, bile flow increased by about 32%, bilirubin content increased by about 28%, and cholesterol content decreased by about 15%. Compared with single use, the combined use had a significant synergistic effect on bile secretion-related indicators, and was superior to the control group.

Claims

1. A small molecule peptide composition for regulating the production of natural bezoar, characterized in that: include: A small molecule peptide Peptide A targeting the novel bile acid transport regulatory protein receptor Target A on hepatocytes, whose amino acid sequence is Glu - Asp - Pro - Thr - Ile; A small molecule peptide Peptide B targeting the specific active region of bilirubin glucuronyltransferase Target B, whose amino acid sequence is His - Val - Met - Arg - Trp; Peptide C is a small molecule peptide targeting Target C, a key node receptor for inflammation regulation on immune cells, and its amino acid sequence is Ser-Lys-Pro-Glu-Phe.

2. A method for preparing the small molecule peptide composition according to claim 1, characterized in that: Using chemical synthesis methods and solid-phase synthesis technology, amino acid monomers are sequentially connected to a solid-phase carrier. After each coupling reaction, a deprotection step is performed to remove the protecting group. Finally, the small molecule peptide is cut from the solid-phase carrier and purified and quality tested.

3. The preparation method according to claim 2, wherein the purification adopts high performance liquid chromatography HPLC and ion exchange chromatography to make the purity of the small molecule peptide reach more than 99.5%.

4. A method for regulating the production of natural bezoar using the small molecule peptide composition of claim 1, characterized in that: The small molecule peptide composition is orally administered to cattle, and the dosage and administration frequency are determined according to the weight, age and health condition of the cattle.

5. The method according to claim 4, wherein for cattle weighing 500-600 kg, the oral dose is 10 mg / kg body weight per day, divided into two doses; the injection dose is once a week, 5 mg / kg body weight each time.