A tripeptide compound, a preparation method thereof and a use of the tripeptide compound for enhancing immune function of a body

Abstract

The application discloses a kind of tripeptide compounds and its preparation method and its machine enhances the use of body immune function, the amino acid sequence of the compound is L-proline-L-threonine-L-tyrosine amide (L-Pro-L-Thr-L-Tyr-NH2), structural formula is, pharmacological experiment result shows, the compound can significantly promote macrophage activation of the application, dose-dependently enhance macrophage phagocytosis, up-regulate the secretion of TNF-alpha, IL-1 beta and NO, by synergistic control macrophage immune function activation innate immune response, with significant immune enhancing activity and new drug development potential.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, specifically relating to a tripeptide compound, its preparation method, and its use in enhancing the body's immune function. Background Technology

[0002] The immune system is the core defense system that protects the body from invading pathogens and maintains homeostasis. Its functional state directly affects the body's health and susceptibility to disease. A strong immune system can not only accurately identify and eliminate pathogenic microorganisms such as bacteria and viruses, reducing the risk of infectious diseases, but also eliminate abnormal cells with gene mutations through immune surveillance, building a strong defense against tumors. Simultaneously, it can regulate the balance of inflammatory responses, preventing excessive inflammation from damaging tissues and organs, and plays a crucial role in wound repair and postoperative recovery. However, against the backdrop of an aging population, widespread use of radiotherapy and chemotherapy for tumors, and an increasing number of postoperative patients, the number of immunocompromised individuals continues to grow. This population often faces higher infection risks, slower recovery processes, and a significantly increased probability of tumor recurrence, necessitating the intervention of safe and effective immune enhancers.

[0003] Currently marketed and research-stage immune enhancers are mainly divided into six categories: cytokines, immune checkpoint inhibitors, microbial-derived preparations, small chemical molecules, natural products, and peptides. Each category has applications in specific scenarios, but all suffer from inherent drawbacks: First, mainstream recombinant cytokines, monoclonal antibodies, thymosin-based macromolecular preparations are easily degraded by gastrointestinal proteases after oral administration, resulting in extremely low bioavailability. Second, small chemical molecule preparations have serious toxic side effects such as bone marrow suppression, teratogenicity, and agranulocytosis. Third, natural product and microbial extract immune enhancers generally suffer from complex compositions and unclear active ingredients.

[0004] Ganoderma lucidum is a fungus belonging to the Polyporaceae family, specifically the red Ganoderma lucidum. Ganoderma lucidum The dried fruiting body of *Leyss. ex Fr.* Karst is a traditional Chinese medicine and food, often referred to as the "immortal herb" in Traditional Chinese Medicine. Its effects of tonifying qi, calming the mind, relieving cough, and alleviating asthma have been clinically verified over a long period. Modern pharmacological research further reveals that *Ganoderma lucidum* plays a significant role in regulating the body's immune function, making it an important representative of natural immune enhancers. Clinically, it is frequently used as an adjunct therapy for patients undergoing radiotherapy and chemotherapy for cancer, as well as for individuals with weakened immune systems. *Ganoderma lucidum* polysaccharides and polypeptides are considered its main active ingredients.

[0005] Peptides, due to their small molecular weight, high bioactivity, and low toxicity, have become an important direction for the research and development of immune enhancers. Compared with Ganoderma lucidum polypeptides, Ganoderma lucidum tripeptides have more prominent advantages: First, higher absorption efficiency. Tripeptides are composed of three amino acids, with a molecular weight much smaller than polypeptides. They can be directly absorbed into the bloodstream from the intestines without undergoing complex digestion and decomposition processes, quickly reaching the target site to exert their effects and effectively improving bioavailability. Second, stronger bioactivity and more precise targeting. The small molecular structure makes tripeptides easier to penetrate cell membranes and bind to specific receptors on the surface of immune cells, resulting in more targeted immunomodulatory activity. Third, higher safety. The metabolic products of small molecule peptides in the body are amino acids, which are easily metabolized and excreted by the body, with no residual risk. Compared with polypeptides with larger molecular weights, they are less likely to cause adverse reactions such as immune rejection or allergies. However, research on Ganoderma lucidum tripeptides is limited. Wu Qiang et al. isolated and purified the Ganoderma lucidum tripeptide Ser-Tyr-Pro from Ganoderma lucidum liquid fermentation broth, which showed significant inhibition of ACE activity. Currently, there are no Ganoderma lucidum-derived tripeptide immune enhancers with a clearly defined structure, definite activity, and fully controllable quality throughout the entire process.

[0006] To address the shortcomings of existing technologies, this invention aims to develop tripeptides with immune-enhancing effects as candidate molecules for immune-enhancing agents, providing more reliable and effective solutions for the intervention of clinical immunodeficiency-related diseases. This invention fills the gap in research and application in this field, and solves the core defects of existing Ganoderma lucidum-based immune enhancers, such as complex composition, poor efficacy reproducibility, and insufficient drug-likeness. It has significant academic value and promising clinical application prospects. Summary of the Invention

[0007] The purpose of this invention is to provide a tripeptide compound that can enhance the body's immune function, and this invention also provides a method for its preparation. Its amino acid sequence is L-proline-L-threonine-L-tyrosine amide (L-Pro-L-Thr-L-Tyr-NH2), and its chemical structure is as follows:

[0008] The method for preparing the tripeptide compound of the present invention includes the following steps: (1) Take Ganoderma lucidum, crush it and pass it through a 200-mesh sieve, soak it at 50℃ to extract it, and filter it to obtain Ganoderma lucidum extract; (2) Preparation of Ganoderma lucidum seed culture: The mycelium of the Ganoderma lucidum mother culture was inoculated onto the slant of PDA slant medium to complete the activation of the strain. The activated Ganoderma lucidum mycelium was then inoculated into Ganoderma lucidum liquid seed culture medium and cultured until a large number of small and uniform mycelial balls were formed. The OD was measured by ultraviolet spectrophotometer. 600 The culture was completed when the OD value was 1.0. The supernatant was then removed by centrifugation. The bacterial precipitate was washed twice with sterile physiological saline, and the cells were resuspended in liquid seed culture medium. The cell concentration was adjusted to OD0.0. 600 =1.0, which means the Ganoderma lucidum seed liquid is obtained; (3) Preparation of Bacillus subtilis seed culture: Bacillus subtilis inoculum was inoculated onto the slant of nutrient agar medium to complete the inoculum activation. The activated bacterial culture was then inoculated into LB liquid medium and cultured until the culture solution was uniformly turbid and the OD value was [value missing]. 600 The culture was completed at an OD value of 0.8. The supernatant was then removed by centrifugation. The bacterial precipitate was washed twice with sterile physiological saline, and the cells were resuspended in LB liquid medium. The bacterial concentration was adjusted to OD. 600 =0.8, which yields Bacillus subtilis seed solution; (4) Preparation of Ganoderma lucidum liquid fermentation broth: Ganoderma lucidum seed broth: Bacillus subtilis seed broth = 2:1 (v / v), mix the two seed broths thoroughly; add the mixed seed broth to the Ganoderma lucidum liquid fermentation medium according to the total volume of the mixed seed broth: Ganoderma lucidum liquid fermentation medium = 1:9 (v / v), and ferment in a deep layer for 7 days. After fermentation, adjust the pH of the fermentation broth to 7.0, add 2% by weight of neutral protease of Ganoderma lucidum raw material powder, and enzymatically hydrolyze for 2 hours. After enzymatic hydrolysis, inactivate the enzyme, then quickly cool to room temperature, filter, and obtain Ganoderma lucidum liquid fermentation enzymatic hydrolysate; (5) Centrifuge the liquid fermentation hydrolysate of Ganoderma lucidum, collect the supernatant, and separate it through 100kDa and 3kDa ultrafiltration membranes in sequence. Collect the filtrate that has passed through the 3kDa ultrafiltration membrane, put it into a 0.1kDa cellulose ester (CE) dialysis bag, and dialyze it with deionized water. After dialysis, collect the solution in the dialysis bag, concentrate it under reduced pressure, and freeze dry it. (6) Take the frozen powder, add deionized water to dissolve it, and use Sephadex G-10 gel column chromatography with deionized water as the elution solvent. Use TLC detection to guide the collection of the eluent. After the molecular weight of the eluent is confirmed by high performance liquid chromatography-mass spectrometry, concentrate it under reduced pressure to a viscous solution to obtain the crude product.

[0009] (7) Take the crude product, dissolve it in deionized water, and further purify it by preparative HPLC. Use a C18 preparative chromatographic column, use 0.1% trifluoroacetic acid-acetonitrile solution as the elution solvent, perform gradient elution, use the ultraviolet detection wavelength of 220 nm, collect the chromatographic peak eluent with a retention time of 27 min, concentrate it and freeze dry it to obtain the compound of the present invention.

[0010] The PDA slant culture medium consists of 200 g / L peeled potato, 20 g / L glucose, 0.5 g / L MgSO4·7H2O, 1 g / L KH2PO4, 20 g / L agar, and the remainder being distilled water.

[0011] The components of the Ganoderma lucidum liquid seed culture medium include 20 g / L glucose, 5 g / L peptone, 3 g / L yeast extract, 1 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, and the remainder being distilled water.

[0012] The components of the nutrient agar slant culture medium include 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 20 g / L agar, and the remainder distilled water.

[0013] The LB liquid culture medium consists of 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and the remainder distilled water.

[0014] The components of the Ganoderma lucidum liquid fermentation culture medium include 20 g / L glucose, 5 g / L peptone, 3 g / L yeast extract, 1 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, and the remainder being Ganoderma lucidum extract.

[0015] In vitro experimental results of the compounds described in this invention show that they can promote macrophage activation in a dose-dependent manner, significantly enhance macrophage phagocytic function, and promote the secretion of IL-1β, TNF-α, and NO by macrophages. They have the effect of enhancing the body's immune function and can be used to prepare immune enhancers. Attached Figure Description

[0016] Figure 1 Results of the effect of the compound of the present invention on the phagocytic capacity of macrophages Figure 2 Results of the effect of the compound described in this invention on TNF-α secretion by macrophages Figure 3 Results of the effect of the compound described in this invention on IL-1β secretion by macrophages Figure 4 Results of the effect of the compound described in this invention on NO secretion by macrophages Figure 5 Nuclear magnetic resonance of the compound described in this invention 1 H-NMR spectrum Figure 6 Nuclear magnetic resonance of the compound described in this invention 1 H-NMR magnification spectrum Figure 7 Nuclear magnetic resonance of the compound described in this invention 1 H-NMR magnification spectrum Figure 8 Nuclear magnetic resonance of the compound described in this invention 1 H-NMR magnification spectrum Figure 9 Nuclear magnetic resonance of the compound described in this invention 13 C-NMR spectrum Figure 10 The HSQC nuclear magnetic resonance spectrum of the compound described in this invention. Figure 11 The nuclear magnetic resonance HMBC spectrum of the compound described in this invention Figure 12 Nuclear magnetic resonance of the compound described in this invention 1 H- 1 H COSY spectrum Figure 13 The HR-ESI-MS spectrum of the compound described in this invention Figure 14 The HPLC chromatograms of the threonine and proline fragments of the compounds described in this invention, as well as the Marfey reagent-derived reactants of L-threonine, D-threonine, L-threonine, and D-threonine reference standards, are shown below. Figure 15 This is a comparison of the tyrosine-NH2CD and ECD spectra of the enzymatic hydrolysis products of the compound described in this invention. Detailed Implementation

[0017] The present invention will be further described below with reference to embodiments, but these embodiments do not limit the implementation of the present invention.

[0018] Example 1: Preparation method of the compound described in this invention (I) Main experimental materials: 1. The Ganoderma lucidum was purchased from Hebei Boaitang Pharmaceutical Co., Ltd.; 2. Ganoderma lucidum strain CGMCC5.616 was purchased from the China General Microbiological Culture Collection Center; 3. Bacillus subtilis ATCC 6633, brand name: Microbiologics; 4. Anhydrous glucose, MgSO4·7H2O, KH2PO4, and sodium chloride were all of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. 5. Peptone, tryptone, yeast extract, and agar powder were all purchased from Beijing Aoboxing Biotechnology Co., Ltd. 6. Neutral protease, derived from Bacillus subtilis, 50,000 U / g, brand name Solarbio; 7. The 100kDa and 3kDa ultrafiltration membranes were purchased from Wuhan Bona New Membrane Materials Co., Ltd. 8. 0.1 kDa cellulose ester (CE) dialysis membrane, brand name: Repligen Spectrum; 9. Sephadex G-10 gel was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; (II) Main Experimental Instruments 1. Constant temperature water bath, HH-S4, Jiangsu Shenglan Instrument Manufacturing Co., Ltd.; 2. Constant temperature shaking incubator, HZQ-F160, Harbin Donglian Electronic Technology Development Co., Ltd.; 3. Preparative HPLC, Shimadzu LC-20AP, Shimadzu; 4. Rotary evaporator, N-1300S-WB, Tokyo RIKEN; 5. Freeze dryer, FDU-1200, Tokyo Rika.

[0019] (III) Experimental Procedure 1. Take the fruiting body of Ganoderma lucidum (red Ganoderma), remove impurities, insect-infested parts and lignified parts of the stipe, crush it through a 200-mesh sieve, add water (w / v) with a weight of 15 times the volume of Ganoderma lucidum powder, soak and extract at a constant temperature of 50℃ for 24 hours, stirring once every 4 hours, and filter to obtain Ganoderma lucidum extract. 2. Preparation of Ganoderma lucidum seed liquid (1) Preparation of PDA slant culture medium: Take 200g of peeled potato, cut it into small pieces, add 0.8L of distilled water and boil for 30min, filter, add 20g of glucose, 0.5g of MgSO·7H2O and 1g of KH2PO4 to the filtrate, stir to dissolve, add distilled water to make up to about 900mL, add 20g of agar, stir constantly, heat to boiling until the agar is completely melted, add distilled water to make up to 1000mL, stir evenly, and dispense into sterile test tubes while hot (about 1 / 4 of the height of the test tube), autoclave at 121℃ for 20min, after sterilization, tilt the test tubes while hot (tilt the angle so that the length of the slant is 2 / 3 of the length of the test tube), cool and solidify completely to make PDA slant culture medium.

[0020] (2) Ganoderma lucidum inoculation and activation culture: Ganoderma lucidum mother culture mycelium is rapidly inoculated onto the slant of PDA slant culture medium and cultured at 28℃ for 7 days until the mycelium covers the slant, thus completing the activation of the culture.

[0021] (3) Preparation of Ganoderma lucidum liquid seed culture medium: Accurately measure a sufficient amount of distilled water, pour it into a beaker, place it on a magnetic stirrer and stir at low speed (50~80 r / min). While maintaining the stirring state, add each nutrient component in sequence: add glucose at a concentration of 20 g / L, add peptone at a concentration of 5 g / L, add yeast extract at a concentration of 3 g / L, add KH2PO4 at a concentration of 1 g / L, and add MgSO4·7H2O at a concentration of 0.5 g / L. Stir thoroughly until completely dissolved, adjust the pH to 6.0 with 1 mol / L NaOH or 1 mol / L HCl, autoclave at 121℃ for 20 min, and cool to room temperature to obtain Ganoderma lucidum liquid seed culture medium.

[0022] (4) Mycelial inoculation and liquid culture: The activated Ganoderma lucidum mycelia were rapidly inoculated into Ganoderma lucidum liquid seed culture medium cooled to room temperature, and cultured at 28℃ and 180r / min for 72h in a constant temperature shaker until a large number of small and uniform mycelial balls were formed. The OD was measured by ultraviolet spectrophotometer. 600The culture was completed when the OD value was 1.0 (the blank control was sterilized Ganoderma lucidum liquid seed culture medium cooled to room temperature). The culture was then centrifuged (8000 rpm, 10 min) to remove the supernatant. The bacterial precipitate was washed twice with sterile physiological saline, and the bacterial cells were resuspended in liquid seed culture medium. The bacterial concentration was adjusted to OD value. 600 =1.0 (the blank control is Ganoderma lucidum liquid seed culture medium that has been sterilized and cooled to room temperature), and the Ganoderma lucidum seed liquid is obtained.

[0023] 3. Preparation of Bacillus subtilis seed culture (1) Preparation of nutrient agar slant culture medium: Accurately measure a sufficient amount of distilled water, pour it into a beaker, place it on a magnetic stirrer and stir at low speed (50~80 r / min). While maintaining the stirring state, add each nutrient component in sequence: add tryptone at a concentration of 10 g / L, yeast extract at a concentration of 5 g / L, sodium chloride at a concentration of 10 g / L, and agar at a concentration of 20 g / L. Stir thoroughly until completely dissolved, adjust the pH to 7.0 with 1 mol / L NaOH or 1 mol / L HCl, heat to boiling to completely melt the agar (stir continuously during heating), and dispense while hot into sterile test tubes (about 1 / 4 of the height of the test tube). Sterilize at 121℃ for 20 min. After sterilization, tilt the test tubes while hot (tilt the angle so that the length of the slant is 2 / 3 of the length of the test tube), and cool to room temperature to obtain the nutrient agar slant culture medium.

[0024] (2) Inoculation and activation culture of Bacillus subtilis strain: Bacillus subtilis strain was rapidly inoculated onto the slant of nutrient agar medium and cultured at 37℃ for 24 hours until a full and uniform bacterial moss grew on the slant, thus completing the activation of the strain.

[0025] (3) Preparation of LB liquid culture medium: Take 10g of tryptone, 5g of yeast extract and 10g of sodium chloride, add about 0.8L of distilled water, stir to dissolve, adjust the pH to 7.0 with 1mol / L NaOH or 1mol / L HCl, add distilled water to make up to 1000mL, autoclave at 121℃ for 20min, cool to room temperature and use.

[0026] (4) Mycelial inoculation and liquid culture: The activated mycelial growth was rapidly inoculated into LB liquid medium and cultured at 37°C with shaking speed of 180 r / min for 12-16 h until the culture medium was uniformly turbid and the OD value was [not specified]. 600 The culture was completed when the OD value was 0.8 (blank control was LB liquid medium cooled to room temperature after sterilization). The culture was then centrifuged (8000 rpm, 10 min) to remove the supernatant. The bacterial precipitate was washed twice with sterile physiological saline, and the cells were resuspended in LB liquid medium. The bacterial concentration was adjusted to OD value. 600=0.8 (the blank control is LB liquid medium that has been sterilized and cooled to room temperature), thus obtaining the Bacillus subtilis seed solution.

[0027] 4. Preparation of Ganoderma lucidum liquid fermentation broth (1) Preparation of Ganoderma lucidum liquid fermentation medium: The prepared Ganoderma lucidum extract was used as the base solvent and poured into a beaker. The beaker was placed on a magnetic stirrer and stirred at low speed (50~80 r / min). While stirring, the following nutrients were added in sequence: glucose at a concentration of 20 g / L, peptone at a concentration of 5 g / L, yeast extract at a concentration of 3 g / L, KH2PO4 at a concentration of 1 g / L, and MgSO4·7H2O at a concentration of 0.5 g / L. The mixture was stirred until completely dissolved. The pH was adjusted to 6.0 with 1 mol / L NaOH or 1 mol / L HCl. The mixture was then sterilized by high-pressure steam at 121℃ for 20 min and cooled to room temperature to obtain the Ganoderma lucidum liquid fermentation medium.

[0028] (2) Accurately measure the two seed liquids according to the ratio of Ganoderma lucidum seed liquid to Bacillus subtilis seed liquid = 2:1 (v / v) and mix them thoroughly. Add the mixed seed liquid to the Ganoderma lucidum liquid fermentation medium according to the ratio of total volume of mixed seed liquid to Ganoderma lucidum liquid fermentation medium = 1:9 (v / v) and ferment in a constant temperature water bath shaker at 30℃ and 180r / min for 7 days. After fermentation, adjust the pH of the fermentation broth to 7.0 with 1mol / L NaOH or 1mol / L HCl, add 2% by weight of neutral protease of Ganoderma lucidum raw material powder (neutral protease needs to be dissolved in sterile physiological saline in advance to prepare a stock solution), stir evenly, and enzymatically hydrolyze at 50℃ for 2 hours. During enzymatic hydrolysis, stir once every 30 minutes. After enzymatic hydrolysis, inactivate the enzyme at 100℃ and normal pressure for 15 minutes, then quickly cool to room temperature in an ice bath, filter, and obtain the Ganoderma lucidum liquid fermentation enzymatic hydrolysate.

[0029] 5. Purification steps (1) Centrifuge the liquid fermentation hydrolysate of Ganoderma lucidum (4℃, 10000r / min, 15min), collect the supernatant, separate it through a 100kDa ultrafiltration membrane, collect the filtrate that has passed through the 100kDa ultrafiltration membrane, separate it through a 3kDa ultrafiltration membrane, collect the filtrate that has passed through the 3kDa ultrafiltration membrane, and put it into a 0.1kDa cellulose ester (CE) dialysis bag. After filling the bag, clamp both ends of the dialysis bag with the dialysis bag clamp, and gently place the sealed dialysis bag into a sterile beaker containing sufficient deionized water. The amount of deionized water should be 10 to 15 times the volume of the solution in the dialysis bag. Dialyze for 24 hours at 4℃ with magnetic stirring (adjust the speed to 50r / min), and replace the deionized water in the beaker every 4 hours. After dialysis, collect the solution in the dialysis bag, concentrate it under reduced pressure at 40℃, and then freeze dry it.

[0030] (2) Take the frozen powder, add deionized water to dissolve it, filter it through a 0.22μm sterile filter membrane, and pass it through Sephadex G-10 gel column chromatography (column inner diameter 3cm, column bed height 90cm, diameter-to-height ratio = 1:30, the volume of the sample solution is 5% of the column bed volume, and the flow rate is 0.3~0.5mL / min). Use deionized water as the elution solvent and collect it once every 5ml in a test tube.

[0031] The eluent from each tube was analyzed by TLC: after appropriate concentration, the sample was spotted onto the activated GF. 254 On a silica gel thin-layer plate, using n-butanol-glacial acetic acid-water (4:1:1, V / V) as the developing solvent, the plate was developed, removed, and dried. It was then examined under a UV lamp (254 nm). The compound described in this invention showed a dark spot at Rf=0.35. After spraying with ninhydrin reagent (2g of ninhydrin dissolved in ethanol to make 100mL), and heating at 105℃, the compound showed a yellow spot at Rf=0.35.

[0032] The target eluents that meet the above characteristics were combined and detected by HPLC-MS. The quasi-molecular ion peak of the target peak had an m / z of 379.2. After verifying the molecular weight, the solution was concentrated under reduced pressure at 40°C to a viscous solution to obtain the crude product.

[0033] (3) Take the crude product, dissolve it in deionized water, filter it with a 0.22 μm sterile filter membrane, and further purify it by preparative HPLC. Chromatographic conditions: C18 preparative column [SHIMADZU Shim-pack GIST (C18, 5 μm, 20 × 250 mm)], with 0.1% trifluoroacetic acid aqueous solution as mobile phase A and acetonitrile as mobile phase B, and perform gradient elution according to the table below; the detection wavelength is 220 nm, the column temperature is 30 °C, and the flow rate is 10 mL / min.

[0034]

[0035] The chromatographic peak eluents with a retention time of 27 min were collected, concentrated under reduced pressure at 40 °C, and then freeze-dried to obtain the compound described in this invention.

[0036] Example 2: Evaluation of the immune-enhancing activity of the compound of the present invention - activation effect on mouse peritoneal macrophages 1. Materials and Instruments (1) Animals: SPF-grade healthy Kunming mice, 6-8 weeks old, weighing 20±2g, half male and half female, purchased from Shandong Aileke Biotechnology Co., Ltd. After purchase, they were placed in a barrier environment with a temperature of 22±2℃ and a humidity of 50±5% for 1 week for acclimatization.

[0037] (2) Reagents The compound described in this invention was prepared in-house and has a content >98%. RPMI-1640 culture medium and fetal bovine serum, brand name Gibco; PBS buffer (pH 7.4, sterile and enzyme-free), trypan blue staining solution, brand: Maclean; Neutral red staining solution (0.1%), brand name Solarbio; LPS (lipopolysaccharide, E.coli O111:B4), brand name Merck; Mouse tumor necrosis factor α (TNF-α) ELISA kit, brand name: MultiSciences; Mouse interleukin 1β ELISA kit, brand name: MultiSciences; Nitric oxide (NO) detection kit (Griess method), brand name MCE.

[0038] (3) Instruments Clean bench: SW-CJ-1F, Suzhou Cleanroom Equipment Co., Ltd.; Air-jacketed CO2 incubator: HF90, Shanghai Likang Instrument Co., Ltd.; High-speed refrigerated centrifuge: TGL-16M, Hunan Xiangyi Laboratory Instrument Development Co., Ltd.; Multifunctional microplate reader: RT-6000, Shenzhen Raydu Life Science Co., Ltd.; Constant temperature water bath: HH-S4, Jiangsu Shenglan Instrument Manufacturing Co., Ltd.

[0039] 2. Experimental Methods (1) Isolation and purification of mouse peritoneal macrophages Mice were euthanized by cervical dislocation. Immediately after disinfection with 75% alcohol, 5 mL of pre-warmed RPMI-1640 culture medium (37°C) without fetal bovine serum was injected intraperitoneally under aseptic conditions. The mice were placed supine and the abdomen was gently massaged for 2-3 minutes. After standing for 5-7 minutes, the abdominal skin was aseptically cut open to expose the peritoneum. When the peritoneal fluid turned pale yellow, approximately 4-4.5 mL of peritoneal fluid was aspirated using a syringe and slowly injected into a sterile centrifuge tube. The tube was centrifuged (1000 rpm, 4°C for 10 minutes), the supernatant was discarded, and the bottom cell pellet was collected. Trypan blue staining was used, and viable cell counting was performed using a hemocytometer. When the viable cell rate reached 95% or higher, the cell concentration was adjusted to 2 × 10⁶ cells / mL with RPMI-1640 culture medium containing 10% fetal bovine serum. 6 Cells / mL were seeded into 96-well cell culture plates, 100 μL per well, and incubated in a CO2 incubator at 37 ℃ with 5% CO2. 2、After culturing for 2 hours under saturated humidity, the original culture medium in the wells was discarded. The well plates were then gently washed twice with sterile PBS buffer preheated to 37°C to thoroughly remove any non-adherent cells. 100 μL of RPMI-1640 medium containing 10% fetal bovine serum was immediately added to each well to obtain purified mouse peritoneal macrophages, which were then used for subsequent drug administration experiments.

[0040] (2) Grouping and administration The cultured macrophages were randomly divided into 5 groups, with 6 replicates per group. All experiments were independently repeated 3 times. The final volume of each well after drug administration was 200 μL. The PBS solvent content was consistent in all groups. The grouping is as follows: Blank control group: Add 100 μL of sterile PBS buffer to each well; Positive control group: Add 100 μL of 10 μg / mL LPS solution (prepared with sterile PBS buffer) to each well. Low-dose sample group: Add 100 μL of the compound solution described in this invention (prepared with sterile PBS buffer) to each well (5 μg / mL). Dosage group in the sample: Add 100 μL of the compound solution described in this invention (prepared with sterile PBS buffer) to each well. High-dose sample group: Add 100 μL of the compound solution described in this invention (prepared with sterile PBS buffer) to each well.

[0041] Each group was incubated at 37℃ in a 5% CO2 incubator for 24 hours. During this period, the culture plates were kept in constant temperature to ensure normal cell growth and full drug effect.

[0042] (3) Macrophage phagocytic capacity detection (neutral red method) After culturing for 24 hours, carefully discard the culture medium and gently wash three times with preheated (37°C) sterile PBS buffer (discard the PBS after each wash to avoid damaging the adherent cells). Add 100 μL of 0.1% neutral red solution (prepared with sterile PBS buffer) to each well and incubate at 37°C for 30 min in a CO2 incubator. Discard the neutral red solution in each well and wash three times with preheated (37°C) sterile PBS buffer to thoroughly remove any unphagocytosed neutral red. Add 100 μL of cell lysis buffer (a 1:1 volume ratio of glacial acetic acid and anhydrous ethanol) to each well and incubate at room temperature for 10 min, gently shaking the culture plate 2-3 times during this period to ensure complete lysis of macrophages and release of phagocytosed neutral red. Measure the absorbance (OD value) of each well at 540 nm using a microplate reader and calculate the phagocytosis rate (phagocytosis rate % = OD value of sample group ÷ OD value of blank control group × 100%).

[0043] (4) Detection of secretion of immune factors TNF-α and IL-1β (ELISA method) After 24 hours of culture, the supernatant from each group was collected using sterile pipettes and centrifuged in sterile centrifuge tubes (3000 rpm, 4°C for 10 min) to remove cell debris. The supernatant was then used to detect TNF-α and IL-1β using ELISA. The procedure was strictly performed according to the kit instructions. The OD value of each well was measured at 450 nm using a microplate reader. Based on the kit's standard curve, the concentrations (pg / mL) of TNF-α and IL-1β in the supernatant of each group were calculated.

[0044] (5) NO secretion detection (Griess method) After 24 hours of culture, the supernatant from each group was collected using sterile pipettes and centrifuged in sterile centrifuge tubes (3000 rpm, 4°C for 10 min) to remove cell debris. NO levels in the supernatant were measured using the Griess method. The procedure was strictly followed according to the kit instructions. The absorbance (OD value) of each well was measured at 540 nm using a microplate reader. The NO content (μmol / L) in the supernatant of each group was calculated based on the standard curve.

[0045] (6) Data Analysis: Experimental data were processed using SPSS 20 statistical analysis software. The data were presented in [data format missing]. ±s indicates that the comparison between groups was performed using one-way ANOVA and the pairwise comparison between groups was performed using the LSD test. P < 0.05 was considered statistically significant.

[0046] 3. Experimental Results (1) The effects of the compound described in this invention on the phagocytic capacity of macrophages are shown in Table 1. Figure 1 From Table 1, Figure 1 As can be seen, the low-dose, medium-dose, and high-dose groups of the compound described in this invention all have a significant enhancing effect on the phagocytic capacity of macrophages. Among them, the phagocytic rate of the medium-dose and high-dose groups of the compound described in this invention is significantly better than that of the LPS group, with the high-dose group being the best.

[0047] Table 1. Effects of the compounds described in this invention on macrophage phagocytic capacity.

[0048] Note: Compared with the blank control group, * indicates P<0.05, ** indicates P<0.01; compared with the LPS group, # This indicates that P < 0.05.

[0049] (2) The effects of the compound described in this invention on the secretion of TNF-α and IL-1β by macrophages are shown in Table 2. Figure 2 , Figure 3 From Table 2, Figure 2As can be seen from Table 2, the low-dose, medium-dose, and high-dose groups of the compound described in this invention all significantly enhanced the secretion of TNF-α by macrophages, with the high-dose group showing significantly better results than the LPS group. Figure 3 As can be seen, the low-dose, medium-dose, and high-dose groups of the compound described in this invention all have a significant effect on increasing the secretion of IL-1β by macrophages, and the high-dose group of the compound described in this invention is significantly better than the LPS group.

[0050] Table 2. Effects of the compounds described in this invention on macrophage secretion of TNF-α and IL-1β.

[0051] Note: Compared with the blank control group, ** indicates P<0.01; compared with the LPS group, # This indicates that P < 0.05.

[0052] (3) The effects of the compound described in this invention on NO secretion by macrophages are shown in Table 3. Figure 4 From Table 3, Figure 4 As can be seen, the low-dose, medium-dose, and high-dose groups of the compound described in this invention all have a significant effect on increasing NO secretion by macrophages, and the high-dose group of the compound described in this invention is significantly better than the LPS group.

[0053] Table 3. Effects of the compounds described in this invention on NO secretion by macrophages.

[0054] Note: Compared with the blank control group, ** indicates P<0.01; compared with the LPS group, # This indicates that P < 0.05.

[0055] In summary, in vitro experiments have confirmed that the compounds described in this invention can significantly promote macrophage activation in a dose-dependent manner; significantly enhance their phagocytic function; and promote the secretion of IL-1β, TNF-α, and NO in a dose-dependent manner.

[0056] Example 3: Structural confirmation of the compound described in this invention This product is a white amorphous powder, soluble in water. High-resolution mass spectrometry yielded an m / z of 379.1976 [M + H]. + The quasi-molecular ion peak (calculated value 379.1976), combined with... 1 H-NMR spectrum and 13 Based on the C-NMR spectral data, the molecular formula of the compound is deduced to be C. 18 H 26 N4O5.

[0057] exist 1The H-NMR spectrum revealed 19 proton signals. In the saturated aliphatic hydrogen region (δ1.0–3.5 ppm): multiple methylene hydrogen signals and one methyl signal, corresponding to the saturated aliphatic chain structure of the amino acid side chain. In the δ3.5–4.8 ppm range: δ4.67, 1H, m (partial signal overlap with solvent peaks), 4.53, 1H, d (J= 7.1 Hz), 4.47, 1H, m, and 3.75, 1H, m, a total of four methylene hydrogen signals connected to N / O. In the aromatic hydrogen region (δ6.5–7.5 ppm): δ7.04, 2H, d (J=8.0 Hz) and δ6.76, 2H, d (J=8.8 Hz), a typical AA'BB' spin system of a para-substituted benzene ring was observed, confirming the presence of one para-substituted benzene ring in the molecule. Additional explanation: The active hydrogen signal disappears due to deuteration in D2O.

[0058] exist 13 The C-NMR spectrum yielded 16 carbon signals. The saturated aliphatic carbon region (δ20–70 ppm) contained 9 carbon signals, including 4 methine carbons (δ68.7, 59.0, 58.0, 55.9 ppm), 4 methylene carbons (δ49.4, 38.6, 30.9, 24.7 ppm), and 1 methyl carbon (δ20.7 ppm), corresponding to the saturated carbon structures of the amino acid skeleton and side chains. The aromatic carbon region (δ115–157 ppm) contained 6 carbon signals: δ156.2 (oxygen-bound quaternary carbon), 130.9 (two overlapping aromatic CH atoms), 129.1 (quaternary carbon), and 115.6 (two overlapping aromatic CH atoms), representing para-substituted benzene rings. The carbonyl carbon region (δ170–175 ppm) contained 3 quaternary carbon signals: δ173.5, 173.0, and 172.1, representing the carbonyl carbons of amides.

[0059] pass 1 H- 1 H COSY, HSQC, and HMBC determined the assignment of three amino acid fragments and their linkage patterns. Tyrosine-NH2 (Tyr-NH2) fragment: C-H assignment was determined using HSQC spectroscopy. In HMBC spectroscopy, δ4.67 (H-2) correlated with δ173.5 (C-1), δ129.1 (C-1'), δ172.1 (C-5), and δ38.6 (C-3); δ3.35 (H-3a) and δ3.16 (H-3b) correlated with δ173.5 (C-1), δ55.9 (C-2), δ129.1 (C-1'), and δ130.9 (C-2' / 6'), thus determining the connection mode of C-1 / C-2 / C-3 / C-5 / C-1' / C-2' / 6'. 1 H-1 In the 1H COSY spectrum, δ4.67 (H-2) correlates with δ3.35 (H-3a) and 3.16 (H-3b), confirming the C-2 / C-3 connection mode; δ7.04 (H-2' / 6') correlates with δ6.76 (H-3' / 5'), verifying the ortho-hydrogen coupling of the benzene ring, consistent with the characteristics of a para-substituted benzene ring.

[0060] Threonine (Thr) fragment: C-H assignment was determined using HSQC spectroscopy. In HMBC spectroscopy, δ4.53 (H-6) correlated with δ173.0 (C-9) and δ20.7 (C-1''); δ4.47 (H-7) correlated with δ172.1 (C-5), δ58.0 (C-6), and δ20.7 (C-1''), confirming the C-5 / C-6 / C-7 / C-1'' / C-9 linkage. 1 H- 1 In the 1H COSY spectrum, δ4.53 (H-6) is correlated with δ4.47 (H-7), and δ4.47 (H-7) is correlated with δ1.25 (H-1''), verifying the C-6 / C-7 / C-1'' linkage, which is consistent with the characteristic threonine skeleton.

[0061] Proline (Pro) fragment: C-H assignment was completed using HSQC spectroscopy. In HMBC spectroscopy, δ3.75 (H-10) correlated with δ173.0 (C-9), δ49.4 (C-1'''), and δ24.7 (C-2'''); δ3.06 (H-1'''a) and δ3.03 (H-1'''b) correlated with δ59.0 (C-10) and δ30.9 (C-3'''); δ2.08 (H-3'''a) and δ1.85 (H-3'''b) correlated with δ173.0 (C-9) and δ49.4 (C-1'''); thus clarifying the connection mode of C-9 / C-10 / C-1''' / C-2''' / C-3'''. 1 H- 1 In the HCOSY spectrum, δ3.75 (H-10) is correlated with δ2.08 (H-3'''a) and δ1.85 (H-3'''b), δ2.08 (H-3'''a) and δ1.85 (H-3'''b) are correlated with δ1.63 (H-2'''), and δ1.63 (H-2''') is correlated with δ3.06 (H-1'''a) and δ3.03 (H-1'''b), forming a closed five-membered ring structure of proline.

[0062] The connection mode of the three amino acid fragments was confirmed: In the HMBC spectrum, δ4.67 (H-2) and δ172.1 (C-5) were correlated, which clarified the peptide bond connection mode of tyrosine and threonine; δ4.53 (H-6) and δ173.0 (C-9) were correlated, which clarified the peptide bond connection mode of threonine and proline.

[0063] In summary, the planar structure of the compound described in this invention is confirmed to be proline-threonine-tyrosine amide (Pro-Thr-Tyr-NH2), and the relevant carbon and hydrogen signal assignments are shown in Table 4.

[0064] Table 4 1 H (600 MHz) and 13 C (150 MHz) NMR Spectroscopic Data in D2O ( δ inppm, J (in Hz)

[0065] Determination of the absolute configuration of the compound described in this invention: An appropriate amount of the product was hydrolyzed in 6 mol / L hydrochloric acid at 110℃ for 24 h. The hydrolysate was evaporated to dryness under reduced pressure, and water was added repeatedly until no hydrochloric acid odor remained. The residue was dissolved in 0.1 mol / L sodium bicarbonate solution, and 1% L-FDAA (Marfey's reagent) acetone solution was added. The mixture was reacted at 40℃ in the dark for 1 h. The reaction was terminated by adding 0.2 mol / L hydrochloric acid, and the solution was diluted to volume with acetonitrile and filtered to obtain the test solution. L-type and D-type proline, threonine, and tyrosine reference standard derivatization solutions were prepared using the same method. A C18 column (Agilent ZORBAX SB-C18, 250 mm × 4.6 mm, 5 μm) was used, with 0.1% trifluoroacetic acid aqueous solution as mobile phase A and acetonitrile as mobile phase B. The detection wavelength was 340 nm. Gradient elution program: 0–45 min, mobile phase A 85% → 45%, mobile phase B 15% → 55%.

[0066] The results showed that the retention times of each amino acid derivative in the test sample were consistent with those of the corresponding L-type amino acid reference standards, and no D-type amino acids were detected, proving that the absolute configurations of proline, threonine, and tyrosine residues in the compound described in the invention are all L-type. Figure 14 .

[0067] The absolute configuration of the tyrosine-NH2 fragment of the compound described in this invention was determined using HPLC-CD coupled with ECD calculation: An appropriate amount of the sample was dissolved and diluted in 100 mmol / L Tris-HCl buffer (containing 10 mmol / L MgCl2, pH 7.8) to prepare a 2 mg / mL solution as the substrate solution. 1 mL of the substrate solution was added to recombinant proline aminopeptidase at a final concentration of 0.5 U / mL, and the reaction was carried out at 37°C in the dark with shaking for 6 h; then, leucine aminopeptidase was added to a final concentration of 1 U / mL, and the reaction was continued at 37°C in the dark with shaking for another 12 h. After the reaction was completed, the pH was adjusted to below 2.0 using 10% trifluoroacetic acid aqueous solution to terminate the reaction. The reaction solution was centrifuged using a 3 kDa ultrafiltration tube to remove the enzyme protein, and the filtrate was used as the test solution.

[0068] HPLC-CD was used for determination using an Agilent ZORBAX SB-C18 column (250 mm × 4.6 mm, 5 μm). The mobile phase was 0.1% trifluoroacetic acid aqueous solution (A) and acetonitrile (B). The detection wavelength was 280 nm. The gradient elution program was: 0–45 min, mobile phase A 85% → 45%, mobile phase B 15% → 55%. Circular dichroism (CD) data were acquired online during the elution time of the tyrosine-NH2 peak, with a detection wavelength range of 200–400 nm.

[0069] Simultaneously, density functional theory (DFT) was used to optimize the conformation of L-tyrosine-NH2 at the B3LYP / 6-311G (d,p) level, and electronic circular dichroism (ECD) calculations were performed. The experimental CD spectrum obtained online was compared with the theoretical ECD spectrum of L-tyrosine-NH2. The peak position, peak shape, and sign direction of the Cotton effect were completely consistent, proving that the absolute conformation of tyrosine-NH2 in this product is L-type. The CD and ECD spectra of the tyrosine-NH2 residues are shown below. Figure 15 .

[0070] In summary, the amino acid sequence of the compound described in this invention is L-proline-L-threonine-L-tyrosine amide (L-Pro-L-Thr-L-Tyr-NH2), and its chemical structure is as follows: .

Claims

1. A tripeptide compound, characterized in that, Its amino acid sequence is L-proline-L-threonine-L-tyrosine amide (L-Pro-L-Thr-L-Tyr-NH2), and its chemical structure is as follows: 。 2. A method for preparing the tripeptide compound according to claim 1, characterized in that: Includes the following steps: (1) Take Ganoderma lucidum, crush it and pass it through a 200-mesh sieve, soak it at 50℃ to extract it, and filter it to obtain Ganoderma lucidum extract; (2) Preparation of Ganoderma lucidum seed culture: The mycelium of the Ganoderma lucidum mother culture was inoculated onto the slant of PDA slant medium to complete the activation of the strain. The activated Ganoderma lucidum mycelium was then inoculated into Ganoderma lucidum liquid seed culture medium and cultured until a large number of small and uniform mycelial balls were formed. The OD was measured by ultraviolet spectrophotometer. 600 The culture was completed when the OD value was 1.

0. The supernatant was then removed by centrifugation. The bacterial precipitate was washed twice with sterile physiological saline, and the cells were resuspended in liquid seed culture medium. The cell concentration was adjusted to OD0.

0. 600 =1.0, which means the Ganoderma lucidum seed liquid is obtained; (3) Preparation of Bacillus subtilis seed culture: Bacillus subtilis inoculum was inoculated onto the slant of nutrient agar medium to complete the inoculum activation. The activated bacterial culture was then inoculated into LB liquid medium and cultured until the culture solution was uniformly turbid and the OD value was [value missing]. 600 The culture was completed at an OD value of 0.

8. The supernatant was then removed by centrifugation. The bacterial precipitate was washed twice with sterile physiological saline, and the cells were resuspended in LB liquid medium. The bacterial concentration was adjusted to OD. 600 =0.8, which yields Bacillus subtilis seed solution; (4) Preparation of Ganoderma lucidum liquid fermentation broth: Ganoderma lucidum seed broth: Bacillus subtilis seed broth = 2:1 (v / v), mix the two seed broths thoroughly; add the mixed seed broth to the Ganoderma lucidum liquid fermentation medium according to the total volume of the mixed seed broth: Ganoderma lucidum liquid fermentation medium = 1:9 (v / v), and ferment in a deep layer for 7 days. After fermentation, adjust the pH of the fermentation broth to 7.0, add 2% by weight of neutral protease of Ganoderma lucidum raw material powder, and enzymatically hydrolyze for 2 hours. After enzymatic hydrolysis, inactivate the enzyme, then quickly cool to room temperature, filter, and obtain Ganoderma lucidum liquid fermentation enzymatic hydrolysate; (5) Centrifuge the liquid fermentation hydrolysate of Ganoderma lucidum, collect the supernatant, and separate it through 100kDa and 3kDa ultrafiltration membranes in sequence. Collect the filtrate that has passed through the 3kDa ultrafiltration membrane, put it into a 0.1kDa cellulose ester (CE) dialysis bag, and dialyze it with deionized water. After dialysis, collect the solution in the dialysis bag, concentrate it under reduced pressure, and freeze dry it. (6) Take the frozen powder, add deionized water to dissolve it, and use Sephadex G-10 gel column chromatography with deionized water as the elution solvent. Use TLC detection to guide the collection of the eluent. After the molecular weight of the eluent is confirmed by high performance liquid chromatography-mass spectrometry, concentrate it under reduced pressure to a viscous solution to obtain the crude product. (7) Take the crude product, dissolve it in deionized water, and further purify it by preparative HPLC. Use a C18 preparative chromatographic column, use 0.1% trifluoroacetic acid-acetonitrile solution as the elution solvent, perform gradient elution, use the ultraviolet detection wavelength of 220 nm, collect the chromatographic peak eluent with a retention time of 27 min, concentrate it and freeze dry it to obtain the compound of the present invention.

3. A method for preparing the tripeptide compound according to claim 2, characterized in that: The PDA slant culture medium consists of 200 g / L peeled potato, 20 g / L glucose, 0.5 g / L MgSO4・7H2O, 1 g / L KH2PO4, 20 g / L agar, and the remainder distilled water.

4. A method for preparing the tripeptide compound according to claim 2, characterized in that: The components of the Ganoderma lucidum liquid seed culture medium include 20 g / L glucose, 5 g / L peptone, 3 g / L yeast extract, 1 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, and the remainder being distilled water.

5. A method for preparing the tripeptide compound according to claim 2, characterized in that: The components of the nutrient agar slant culture medium include 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 20 g / L agar, and the remainder distilled water.

6. A method for preparing the tripeptide compound according to claim 2, characterized in that: The LB liquid culture medium consists of 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and the remainder distilled water.

7. A method for preparing the tripeptide compound according to claim 2, characterized in that: The components of the Ganoderma lucidum liquid fermentation culture medium include 20 g / L glucose, 5 g / L peptone, 3 g / L yeast extract, 1 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, and the remainder being Ganoderma lucidum extract.

8. A tripeptide compound according to claim 1, characterized in that: It can promote macrophage activation in a dose-dependent manner, significantly enhance macrophage phagocytic function, and promote the secretion of macrophage IL-1β, TNF-α and NO.

9. A tripeptide compound according to claim 1, characterized in that: It has the effect of enhancing the body's immune function and can be used to prepare immune enhancers.

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