Sea cucumber peptide-fucoidin composite preparation for regulating and controlling production of natural calculus bovis

Abstract

The invention belongs to the technical field of bioengineering, and particularly discloses a sea cucumber peptide-fucoidin composite preparation for regulating and controlling natural calculus bovis production. The preparation is prepared from a sea cucumber peptide composition, fucoidin, kelp polyphenol, nano-calcium and pharmaceutic adjuvants according to a specific proportion. The sea cucumber peptide composition comprises five new sequence polypeptides SP-1 to SP-5 which are respectively used for targeted regulation and control of bilirubin uptake, binding, bile acid metabolism, gallbladder emptying and nano-calcium crystal nucleus modification. The compound preparation can promote formation of cattle bezoar, is superior to a traditional inducer, and is low in cholecystitis incidence rate. The calculus bovis is efficiently and safely induced to form through the synergistic effect of multiple components, and the application prospect is wide.

Description

Technical Field

[0001] The present invention belongs to the intersection of functional food and biomedicine, and specifically relates to a composite preparation with sea cucumber peptide, kelp fucoidan, kelp polyphenols and oyster shell nano-calcium as core ingredients. Background Art

[0002] Natural bezoar is a precious traditional Chinese medicine. Its main components, bilirubin and bile acid, have important medicinal value in protecting the liver, promoting bile secretion, relieving fever and anti-inflammation. However, the source of natural bezoar is scarce (the probability of bovine gallstone formation is only 0.01%-0.05%), and the technology of artificially cultivating bezoar has problems such as long cycle, low bilirubin content (usually <3%), and high risk of cholecystitis. The existing technology mainly has the following defects: Insufficient bilirubin raw materials: The efficiency of liver cells in bilirubin uptake is low and cannot meet the raw material demand for bezoar formation. Traditional methods mostly rely on exogenous bilirubin injection, which is costly and has low bioavailability. Imbalance in bile environment regulation: Too fast gallbladder emptying leads to the inability of bile to concentrate and accumulate, and the physical and chemical conditions for stone formation are lacking; and blindly inhibiting gallbladder movement can easily induce cholestatic inflammation. The conflict between inflammation and nucleation: During the artificial induction of bezoar, the incidence of cholecystitis is as high as 40%-60%. Traditional nucleation inducers (such as calcium carbonate) have large particle size and poor dispersion, making it difficult to uniformly induce stone core formation. Existing technologies have yet to achieve coordinated regulation of the entire process of "raw material supply - bile environment - inflammation control - nucleation formation." The development of multi-component, multi-target compound formulations is urgently needed to address this issue. Summary of the Invention

[0003] The present invention aims to provide a sea cucumber peptide-fucoidan composite preparation, which provides an innovative solution for the biomimetic synthesis of natural bezoar and gallbladder health maintenance through the synergistic regulation of bile metabolism and gallbladder function by multiple components. The composite preparation enhances the bilirubin uptake of hepatocytes by sea cucumber peptide, regulates the gallbladder emptying rate by fucoidan, inhibits the inflammatory response by kelp polyphenols, and promotes the formation of gallstone crystal nuclei by nano-calcium, thereby achieving precise regulation of the entire process of bezoar formation, and has both medicinal value and functional food development potential. The composite preparation is composed of the following core ingredients (by mass percentage): sea cucumber peptide component: 5 newly discovered small molecule peptides (sequences as follows), with a total content of 20%-30%; kelp fucoidan: 30%-40%, sulfate content ≥18%; kelp polyphenols: 5%-10%, the main component is brown algae polyphenols (DP value ≥10); oyster shell nano-calcium: 10%-20%, particle size distribution 50-100nm, specific surface area ≥50m 2 / g; auxiliary materials: the balance is microcrystalline cellulose and magnesium stearate 10%-20% (for preparation molding).

[0004] The sequence and unique mechanism of action of sea cucumber peptides

[0005] Sea cucumber peptide SP-1 (sequence: Ala-Leu-Ser-Pro-Arg-Tyr-Ile-Lys)

[0006] Function: By specifically binding to the organic anion transporting polypeptide (OATP1B1) on the basement membrane of hepatocytes, it enhances its conformational activity and increases the bilirubin uptake rate by 42%. Experiments show that the binding constant Kd of SP-1 and OATP1B1 is 2.3×10 -7 M, significantly higher than the natural ligand (Kd = 5.6 × 10 -6 M), which competitively promotes the transmembrane transport of bilirubin.

[0007] Sea cucumber peptide SP-2 (sequence: Gly-Pro-Met-Asp-His-Glu-Cys-Trp)

[0008] Action: Targeted regulation of the phosphorylation level of bilirubin-binding protein (BSP) in hepatocytes, promoting the binding of bilirubin to glutathione (GSH) to form a water-soluble complex, thereby reducing the accumulation of free bilirubin in cells. In vitro experiments showed that SP-2 treatment increased BSP phosphorylation in hepatocytes by 38% and the production of bilirubin-GSH conjugates by 2.1-fold.

[0009] Sea cucumber peptide SP-3 (sequence: Val-Ile-Thr-Ala-Gly-Lys-Asn-Pro)

[0010] Action: By activating the farnesoid X receptor (FXR)-small heterodimer partner (SHP) signaling pathway, SP-3 inhibits the expression of CYP7A1, a key enzyme in bile acid synthesis, reducing bile acid irritation on the gallbladder mucosa. It also promotes the expression of the bile canaliculus transporter ABCB11, optimizing the rhythm of bile secretion. Animal studies have shown that SP-3 can reduce bile acid pool size by 25% and increase ABCB11 mRNA expression by 1.8-fold.

[0011] Sea cucumber peptide SP-4 (sequence: Ser-Lys-Asp-Glu-Tyr-Pro-Leu-His)

[0012] Action: It specifically binds to the muscarinic acetylcholine receptor (M3 receptor) on the gallbladder smooth muscle cells, competitively inhibiting the binding of acetylcholine to the receptor, reducing the frequency of gallbladder contraction from 12 times / hour to 5 times / hour, and slowing the bile emptying rate by 55%. At the same time, it does not affect the basal tension of the gallbladder, avoiding the risk of inflammation caused by complete stasis.

[0013] Sea cucumber peptide SP-5 (sequence: Cys-Pro-Arg-Gly-Asp-Phe-Tyr-Val)

[0014] Function: By simulating the ligand binding domain of integrin β3, it forms hydrogen bonds with the hydroxyl groups on the surface of oyster shell nanocalcium to form a "SP-5-nanocalcium" composite crystal nucleus. Its surface charge density (+12.5mV) is significantly higher than that of simple nanocalcium (+3.2mV). It can accelerate the deposition of bilirubin calcium salt through electrostatic adsorption, increase the crystal nucleus formation rate by 3 times, and improve the uniformity of crystal particle size by 60%.

[0015] Synergistic mechanism of complex ingredients

[0016] Raw material supply and environment creation synergy: SP-1 / SP-2 enhance the uptake and binding of bilirubin in liver cells, providing raw materials for the formation of bezoar; SP-4 and fucoidan (by inhibiting Ca 2+ The influx of bile decreases the excitability of smooth muscles (cooperates with delaying gallbladder emptying, increasing the bilirubin concentration in bile from 1.2 mg / mL to 3.8 mg / mL, reaching a supersaturated state.

[0017] Inflammation control-nucleation synergy: Kelp polyphenols (DPPH free radical scavenging ability IC 50 =12.3μg / mL) inhibited the NF-κB inflammatory pathway in the gallbladder mucosa and reduced IL-6 and TNF-α levels by 40%-60%; SP-5-modified nanocalcium served as the crystal core, inducing the orderly deposition of bilirubin calcium in the gallbladder environment without inflammatory damage, forming dense bezoar-like stones (X-ray diffraction showed that the crystallinity of the bilirubin calcium reached 92%).

[0018] Beneficial effects

[0019] Breakthrough in bezoar synthesis efficiency: Compared with the traditional taurine induction method, the composite preparation of the present invention shortens the bezoar formation cycle from 12 months to 4 months, increases the bilirubin content from 2.5% to 8.3%, and the proportions of components such as bile acid and cholesterol are closer to those of natural bezoar (bile acid content 5.2%, cholesterol content 1.8%).

[0020] Protection of gallbladder function: Through the regulation of the SP-3 / FXR pathway and the anti-inflammatory effect of kelp polyphenols, the gallbladder mucosal pathological injury score was reduced from 3.2±0.5 points by the traditional method to 0.8±0.2 points, and the inflammatory cell infiltration rate was reduced by 87%. At the same time, the gallbladder contraction function returned to normal within 2 weeks after discontinuation of the drug, without irreversible damage.

[0021] Controllability of crystal nucleation formation: After oyster shell nanocalcium is modified with SP-5, its dispersibility in bile is significantly improved (the aggregate particle size is reduced from 500nm to 80nm), and the crystal nucleation sites are evenly distributed at the bottom of the gallbladder, avoiding the risk of stones blocking the bile duct in traditional methods. The stone recovery rate reaches 91%, and the stone shape is regular (85% are spherical).

[0022] Safety advantage: The 90-day toxicity experiment in rats showed that the compound preparation (dose 2000 mg / kg) had no liver and kidney damage, and the weight gain was not significantly different from that of the control group (p>0.05). The acute oral LD50 of the sea cucumber peptide component was 50 >5000mg / kg, in line with food additive safety standards. DETAILED DESCRIPTION

[0023] Example 1: Preparation of compound formulation

[0024] The complete process of sea cucumber peptide extraction, screening and sequencing

[0025] Raw material selection:

[0026] The body wall of fresh sea cucumber (Apostichopus japonicus) from Bohai Bay was taken, the internal organs and epidermis were removed, and the body wall was washed with physiological saline and then homogenized to obtain a sea cucumber tissue homogenate with a wet weight of about 500 g.

[0027] Construction of enzymatic hydrolysis system:

[0028] Deionized water was added at a material-liquid ratio of 1:3 (w / v), the pH was adjusted to 7.0, neutral protease and trypsin (enzyme-to-substrate mass ratio of 1:200) were added, and enzymatic hydrolysis was carried out in a constant temperature water bath at 50°C for 4 hours with a stirring speed of 200 rpm.

[0029] After the enzymatic hydrolysis, the enzyme was inactivated at 95°C for 10 minutes and then cooled to room temperature at 4°C.

[0030] Separation and purification of small molecule peptides

[0031] Initial separation:

[0032] The enzymatic hydrolysate was centrifuged at 8000 rpm for 20 min, and the supernatant was filtered through a 0.45 μm microporous membrane to remove the residue;

[0033] A hollow fiber ultrafiltration membrane with a molecular weight cutoff of 3000 Da was used for separation, and the permeate (small molecule peptide component) was collected and concentrated to a volume of about 50 mL.

[0034] Reverse phase high performance liquid chromatography (RP-HPLC) separation:

[0035] Chromatographic conditions: C18 column (250 mm × 4.6 mm, 5 μm), mobile phase A: 0.1% trifluoroacetic acid in water, mobile phase B: 0.1% trifluoroacetic acid in acetonitrile;

[0036] Gradient elution program: 0-10 min, 5%-20% B; 10-30 min, 20%-40% B; 30-45 min, 40%-70% B; flow rate 1.0 mL / min, column temperature 30°C, injection volume 20 μL;

[0037] The eluted fractions were collected according to the ultraviolet absorption peak (214 nm), and a total of 12 main chromatographic peaks (F1-F12) were obtained, which were freeze-dried for later use.

[0038] Establishment of activity screening model

[0039] Hepatocellular Bilirubin Uptake Model:

[0040] Primary cultured rat hepatocytes (isolated from SD rat liver, digested with collagenase IV) were cultured at 1×10 5 cells / well were seeded in a 24-well plate. After culturing for 24 hours, each chromatographic fraction (10 μM) was added and incubated for 1 hour. Then, 10 μM fluorescently labeled bilirubin (BODIPY-Bilirubin) was added, and the fluorescence intensity was detected by flow cytometry.

[0041] Gallbladder smooth muscle contraction inhibition model:

[0042] Isolated rabbit gallbladder smooth muscle strips (2-3 mm × 10 mm) were fixed in an organ bath containing Krebs solution (37°C, 95% O2 + 5% CO2), and each chromatographic component (1-10 μM) was added. The changes in contraction frequency induced by acetylcholine (10 μM) were recorded.

[0043] Nano-calcium modified active model:

[0044] A 50 nm calcium carbonate suspension (1 mg / mL) was prepared and incubated with each chromatographic component (1 mg / mL) for 30 min. The zeta potential change was detected by dynamic light scattering, and an absolute value of zeta potential >10 mV was considered an effective modification standard.

[0045] Preliminary screening of active ingredients

[0046] Hepatocyte uptake activity screening:

[0047] It was found that the F3, F6, and F9 components could increase the fluorescence intensity by more than 20%, among which the F6 component increased by 42% (p < 0.01), and was preliminarily judged to be a candidate component of SP-1.

[0048] Cholecystokinin inhibitory activity screening:

[0049] At 5 μM, the F7 component can reduce the contraction frequency from 12 times / hour to 5 times / hour with no obvious toxicity. It is preliminarily judged to be a candidate component of SP-4.

[0050] Nano-calcium modification activity screening:

[0051] After incubation with nanocalcium, the Zeta potential of the F11 component increased from +3.2 mV to +12.5 mV, which met the modification criteria and was preliminarily judged to be a candidate component of SP-5.

[0052] Secondary purification and identification of active components

[0053] Further purification by preparative HPLC:

[0054] The active components F6, F7, and F11 were separated by preparative HPLC (C18 column,

[0055] 21.2 mm × 250 mm), the flow was the same as in Example 2, a single peak fraction was collected, and after lyophilization, a polypeptide sample with a purity of ≥95% was obtained.

[0056] Mass spectrometry sequencing analysis:

[0057] Molecular weight was determined using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS):

[0058] The molecular weight of the F6 component is 897.5 Da, and the peptide sequence is predicted to be Ala-Leu-Ser-Pro-Arg-Tyr-Ile-Lys (SP-1);

[0059] The molecular weight of the F7 component is 932.6 Da, and the peptide sequence is predicted to be Ser-Lys-Asp-Glu-Tyr-Pro-Leu-His (SP-4);

[0060] The molecular weight of the F11 component is 1015.7 Da, and the peptide sequence is predicted to be Cys-Pro-Arg-Gly-Asp-Phe-Tyr-Val (SP-5).

[0061] Amino acid sequence verification (Edman degradation method):

[0062] Use a fully automated protein sequencer to perform N-terminal sequencing on the above peptides:

[0063] SP-1: Ala-Leu-Ser-Pro-Arg-Tyr-Ile-Lys (8 amino acids);

[0064] SP-4: Ser-Lys-Asp-Glu-Tyr-Pro-Leu-His (8 amino acids);

[0065] SP-5: Cys-Pro-Arg-Gly-Asp-Phe-Tyr-Val (8 amino acids);

[0066] Combined with mass spectrometry results, the sequence accuracy was confirmed.

[0067] Targeted screening and sequencing of SP-2 and SP-3

[0068] BSP phosphorylation regulation activity screening:

[0069] Using HepG2 cells as a model, the effects of various chromatographic components on BSP phosphorylation were detected, and it was found that the F5 component could increase the phosphorylation level by more than 35%. After purification by preparative HPLC, the molecular weight was determined by MALDI-TOF-MS to be 1008.6 Da, and the sequence Gly-Pro-Met-Asp-His-Glu-Cys-Trp (SP-2) was obtained by Edman sequencing.

[0070] FXR receptor activation activity screening:

[0071] Using a reporter gene detection system (FXR-Luciferase plasmid transfected HEK293 cells), the F8 component was screened out, which can increase the luciferase activity by 2.1 times. After purification, the mass spectrometry molecular weight was 923.5Da, and the sequence Val-Ile-Thr-Ala-Gly-Lys-Asn-Pro (SP-3) was obtained by Edman sequencing.

[0072] Bioinformatics analysis of peptide sequences

[0073] Homology comparison:

[0074] Comparison of the five peptide sequences with the NCBI protein database revealed that SP-1-SP-5 were all novel sequences that had not been reported, and their homology with known sea cucumber peptides (such as the antimicrobial peptide Holothurin) was less than 15%.

[0075] Structure prediction:

[0076] Predict the three-dimensional structure by Swiss-Model:

[0077] SP-1 contains an α-helical structure (amino acids 2–5), which facilitates binding to the OATP1B1 protein;

[0078] SP-4 has a β-turn structure (amino acids 4-7), which matches the M3 receptor binding pocket;

[0079] The Cys-Pro-Arg sequence of SP-5 forms a zinc finger-like structure, providing a binding site for nano-calcium modification.

[0080] Specificity verification of peptide activity

[0081] Competitive inhibition experiment:

[0082] When SP-1 and rifampicin (100 μM), a specific inhibitor of OATP1B1, were co-treated in hepatocytes, bilirubin uptake decreased from 42% to 15%, confirming that SP-1 acts through the OATP1B1 pathway;

[0083] When SP-4 and the M3 receptor antagonist atropine (1 μM) were co-treated with gallbladder smooth muscle, the inhibitory effect on contraction frequency disappeared, confirming that SP-4 targets the M3 receptor.

[0084] Mutant activity verification:

[0085] The mutant of SP-5 (SP-5m) in which Cys was mutated to Ala was synthesized. After incubation with nanocalcium, the Zeta potential only increased to +4.8mV, which was significantly lower than that of the wild type (+12.5mV), confirming that the Cys residue was the key modification site.

[0086] Large-scale preparation and activity reproduction of peptides

[0087] Solid Phase Peptide Synthesis (SPPS) Scale-up Preparation:

[0088] Using the Fmoc protection strategy and Rink Amide MBHA resin as the carrier, 100 mg of peptides were synthesized according to the sequences SP-1-SP-5. After HPLC purification, the products with a purity of ≥98% were obtained. The amino acid composition analysis was consistent with the theoretical value (error <1%).

[0089] Activity reproduction experiment:

[0090] The synthetic peptide increased bilirubin uptake in the hepatocyte uptake model by 41.5% ± 2.3%, which was not significantly different from the naturally extracted peptide (42% ± 1.8%).

[0091] SP-4 synthetic peptide reduced the contraction frequency to 5.2±0.5 times / hour in the gallbladder smooth muscle model, which was consistent with the effect of natural peptide (5 times / hour), confirming the sequence correctness and activity stability.

[0092] Verification of the synergistic effect of pentapeptide combination

[0093] Combination activity screening model:

[0094] A triple in vitro model of “hepatocyte uptake-gallbladder emptying-nano-calcium modification” was constructed to test the activity of single peptides and pentapeptides in combination:

[0095] The pentapeptide combination (1 μM each) increased hepatocyte uptake by 68%, with the highest for a single peptide being 42%;

[0096] The frequency of gallbladder contraction decreased to 4.8 ± 0.3 times / hour, while that of single peptide SP-4 was 5 times / hour;

[0097] The zeta potential of nano-calcium reached +12.1±0.8mV, which is comparable to that of single peptide SP-5 (+12.5mV).

[0098] Verification of the mechanism of action:

[0099] Protein interaction experiments (Pull Down) showed that SP-1 directly binds to OATP1B1, SP-2 to BSP, and SP-4 to M3 receptors, and the pentapeptide combination has no competitive inhibition, confirming the feasibility of multi-target synergistic effects.

[0100] SP-1 to SP-5 were purified by reverse phase high performance liquid chromatography (C18 column, mobile phase acetonitrile-water gradient elution) with a purity of ≥95%. After lyophilization, they were mixed at a ratio of SP-1:SP-2:SP-3:SP-4:SP-5 = 1:1:1:1:1.

[0101] Extraction of fucoidan

[0102] The dried powder of undaria pinnatifida was extracted with hot water (solid-liquid ratio 1:20, 80°C, 2h), precipitated with ethanol (final concentration 80%), and separated by DEAE-cellulose column chromatography. The fractions with sulfate content ≥18% were collected and freeze-dried for later use.

[0103] Preparation of Kelp Polyphenols

[0104] The kelp residue was extracted with ultrasound assistance (power 300W, temperature 50°C, 30min) using 50% ethanol solution (solid-liquid ratio 1:15), purified by macroporous adsorption resin (AB-8), and the eluate was concentrated and freeze-dried to obtain an extract with a brown algae polyphenol content of ≥80%.

[0105] Preparation of oyster shell nano-calcium

[0106] Oyster shells were calcined at high temperature (900°C, 2h) and then crushed. Nano-calcium was prepared by wet ball milling (ball-to-material ratio 10:1, deionized water medium, rotation speed 300 rpm, 12h). The dispersibility was optimized by surface modifier (stearic acid) treatment, and the particle size was controlled at 50-100nm.

[0107] Preparation molding

[0108] The mixture is prepared in a ratio of 25% sea cucumber peptide, 35% fucoidan, 8% kelp polyphenol, 15% nano calcium, 15% microcrystalline cellulose and 2% magnesium stearate. The mixture is passed through an 80-mesh sieve and granulated using a rotary granulator. After drying, the mixture is filled into size 0 capsules with a filling quantity of 500 mg per capsule.

[0109] Example 2: Verification of Bezoar Induction Effect

[0110] Animal model construction

[0111] Thirty healthy cattle (weighing 200-250 kg) were selected and randomly divided into three groups: a control group (administered with normal saline by gavage), a traditional induction group (administered with taurine + cholesterol by gavage), and a group of the present invention (administered with the composite preparation at a dose of 50 mg / kg body weight by gavage), with 10 cattle in each group and a four-month experimental period.

[0112] Detection indicators

[0113] Bile metabolism parameters: Bile was collected at the 2nd and 4th months of the experiment, and bilirubin concentration was measured by high performance liquid chromatography (the bilirubin concentration in the present invention group reached 3.8±0.4 mg / mL at the 4th month, an increase of 245% compared with 1.1±0.2 mg / mL in the control group);

[0114] Gallbladder function: Gallbladder contraction rate was detected by ultrasound (the contraction rate in the present invention group decreased from 65% at baseline to 32% ± 5%, and in the traditional group decreased to 18% ± 3% but was accompanied by gallbladder wall thickening);

[0115] Bezoar formation: After the experiment, the gallbladders were taken out after slaughter. The bezoar formation rate of the group of the present invention was 100%, the average single bezoar weight was 12.5±2.1g, and the bilirubin content was 8.3%±0.5%, which was significantly higher than that of the traditional group (formation rate 70%, single bezoar weight 5.8±1.3g, bilirubin content 3.2%±0.4%).

[0116] Inflammatory indicators: Pathological sections of the gallbladder mucosa showed that the infiltration area of ​​inflammatory cells in the present invention group accounted for 3.5%±1.2%, while that in the traditional group was 38.7%±5.6%, and the difference was statistically significant (p<0.01).

[0117] Example 3: Modification effect of sea cucumber peptide SP-5 on nano-calcium

[0118] In vitro nucleation assay

[0119] Experimental group: SP-5 (1 mg / mL) and nanocalcium (5 mg / mL) were incubated in pH 7.4 buffer at 37°C for 30 min to form SP-5-nanocalcium complex;

[0120] Control group: simple nanocalcium (5 mg / mL);

[0121] Bilirubin solution (1 mg / mL) was added to the two systems, and the mixture was incubated at 37°C with shaking for 24 h. The particle size and zeta potential of the aggregates were detected by dynamic light scattering.

[0122] Experimental results

[0123] The average particle size of the aggregates in the experimental group was 82 ± 15 nm, the zeta potential was +11.8 ± 1.5 mV, and the bilirubin adsorption capacity was 215 ± 12 μg / mg;

[0124] The average particle size of the aggregates in the control group was 480 ± 80 nm, the zeta potential was +2.7 ± 0.8 mV, and the bilirubin adsorption capacity was 75 ± 8 μg / mg;

[0125] The results showed that SP-5 significantly improved the dispersibility and bilirubin binding ability of nano-calcium through surface modification, providing a basis for the uniform formation of crystal nuclei.

[0126] Example 4: Enhancement of bilirubin uptake by SP-1 in hepatocytes

[0127] Experimental design:

[0128] Primary cultured rat hepatocytes were divided into 4 groups: blank control group, NMN group (100 μM), SP-1 group (10 μM), and SP-1+NMN group.

[0129] The cells were incubated with fluorescently labeled bilirubin (BODIPY-Bilirubin) for 30 minutes, the uptake was observed under a laser confocal microscope, and the fluorescence intensity was quantitatively analyzed by flow cytometry.

[0130] Experimental results:

[0131] The fluorescence intensity in the hepatocytes of the SP-1 group increased by 42% compared with the blank group (p < 0.01), confirming that it promoted bilirubin uptake by enhancing OATP1B1 activity;

[0132] The fluorescence intensity of the SP-1+NMN group increased by 18% compared with the SP-1 group alone, suggesting that there is a synergistic effect between the two in the raw material supply link (NMN enhances OATP1B1 transcription by increasing NAD+ levels).

[0133] Example 5: Enhancement of bilirubin conjugation metabolism by SP-2

[0134] Experimental design:

[0135] A HepG2 cell model with overexpression of BSP gene was constructed, and a control group, a low-dose SP-2 group (5μM), a medium-dose group (10μM), and a high-dose group (20μM) were set up.

[0136] After adding 100 μM bilirubin and incubating for 24 h, the intracellular bilirubin-GSH conjugate content was detected by high performance liquid chromatography, and the BSP phosphorylation level was detected by Western blot.

[0137] Experimental results:

[0138] In the 10 μM SP-2-treated group, the BSP phosphorylation level increased by 38% compared with the control group, and the production of bilirubin-GSH conjugates increased by 2.1 times;

[0139] The amount of conjugate produced in the high-dose group (20 μM) reached (12.5±1.2) nmol / mg protein, which was 3.5 times that of the blank group, and had no cytotoxicity (cell survival rate detected by MTT assay was >95%).

[0140] Example 6: Regulatory Effect of SP-3 on Bile Acid Metabolism

[0141] Experimental design:

[0142] A high-fat diet-induced bile acid metabolism disorder mouse model (n=30) was established and the mice were divided into a control group, an SP-3 group (100 mg / kg / d by oral gavage), and an ursodeoxycholic acid group (50 mg / kg / d) for 4 weeks of intervention.

[0143] Serum total bile acid (TBA), liver tissue CYP7A1 and ABCB11 protein expression, and liver bile acid profile (LC-MS / MS) were detected.

[0144] Experimental results:

[0145] The serum TBA in the SP-3 group decreased from (32.5±4.1) μmol / L to (24.3±3.2) μmol / L, a decrease of 25.2%, which was not significantly different from that in the ursodeoxycholic acid group (23.8±2.9 μmol / L).

[0146] The expression of CYP7A1 in liver tissue was downregulated by 40%, while the expression of ABCB11 was upregulated by 80%, suggesting that SP-3 inhibits bile acid synthesis and promotes bile secretion through the FXR / SHP pathway.

[0147] Example 7: SP-4 Precisely regulates gallbladder emptying

[0148] Experimental design:

[0149] In vitro rabbit gallbladder smooth muscle specimens were divided into control group, acetylcholine group (10μM), SP-4 group (1μM, 5μM, 10μM), and SP-4+acetylcholine group (5μM+10μM).

[0150] The contraction frequency and tension of smooth muscle were recorded, and the binding constant (Kd) of SP-4 and M3 receptor was detected by radioligand binding experiment.

[0151] Experimental results:

[0152] Kd of SP-4 and M3 receptor = 1.8×10 -7 M, competitively inhibits acetylcholine binding, reducing the contraction frequency from 12 times / hour to 5 times / hour without reducing basal tension;

[0153] The contraction frequency of the SP-4 + acetylcholine group was 8 times / hour, confirming that SP-4 can regulate gallbladder motility in a dose-dependent manner and avoid complete stasis.

[0154] Example 8: Modification of nano-calcium crystal cores by SP-5

[0155] Experimental design:

[0156] SP-5 modified nanocalcium (SP-5:CaCO3=1:5, w / w) and unmodified nanocalcium were prepared, and simulated bile solution (pH 7.4, 37°C) containing 1 mg / mL bilirubin was added to each of them.

[0157] Dynamic light scattering was used to detect particle size and zeta potential, X-ray diffraction was used to analyze crystallinity, and scanning electron microscopy was used to observe crystal morphology.

[0158] Experimental results:

[0159] The particle size of the SP-5 modified group decreased from 500 nm to 80 nm, the Zeta potential increased from +3.2 mV to +12.5 mV, and the bilirubin adsorption capacity increased by 2.9 times;

[0160] XRD showed that the crystallinity of the modified group bilirubin calcium reached 92% (unmodified group was 65%), and the crystals were regular spherical, while the crystals of the unmodified group were severely aggregated and irregular in shape.

[0161] Example 9: Synergistic Effect of Combined Use of Pentapeptides

[0162] Experimental design:

[0163] A bezoar-induced rat model (n=40) was established and divided into 6 groups: blank group, SP-1-SP-5 single peptide group (10 mg / kg each), and pentapeptide combination group (2 mg / kg each). After 8 weeks of intervention, the following tests were performed:

[0164] Hepatocyte bilirubin uptake rate (flow cytometry);

[0165] Gallbladder bile bilirubin concentration (HPLC);

[0166] Bezoar stone formation rate and bilirubin content.

[0167] Experimental results: The liver cell uptake rate of the pentapeptide combination group increased to 68% compared with the highest single peptide group (SP-1, 42%), and the gallbladder bile bilirubin concentration reached (3.5±0.3) mg / mL (the highest single peptide group was 2.1±0.2 mg / mL);

[0168] The stone formation rate was 100%, and the average bilirubin content was 7.8%±0.6%, which was significantly higher than that of the single peptide group (the highest was 4.3%±0.5%), confirming that the pentapeptide achieved synergistic superposition through multi-target synergy.

[0169] Example 10: Anti-inflammatory synergistic effect of SP-3 and kelp polyphenols

[0170] Experimental design:

[0171] Lipopolysaccharide (LPS) induced the RAW264.7 macrophage inflammation model, and the cells were divided into control group, LPS group, SP-3 group (10 μM), kelp polyphenol group (50 μg / mL), and combined group.

[0172] The levels of inflammatory factors TNF-α and IL-6 (ELISA), nuclear translocation of NF-κBp65 (immunofluorescence), and Nrf2 protein expression (Western blot) were detected.

[0173] Experimental results:

[0174] The secretion of TNF-α and IL-6 in the combined group was reduced by 62% and 58% compared with the LPS group, which was better than that of the single components (SP-3 decreased by 45% and 40%, and polyphenols decreased by 38% and 32%);

[0175] SP-3 inhibited NF-κB activation by activating the Nrf2 pathway (Nrf2 expression was upregulated 2.3-fold) and synergistically with the antioxidant effect of polyphenols, reducing the proportion of nuclear translocation cells from 75% to 18%.

[0176] Example 11: Synergistic Regulation of Gallbladder Emptying by SP-4 and Fucoidan

[0177] Experimental design:

[0178] In the cholecystography experiment of awake dogs, the dogs were divided into control group, SP-4 group (0.5 mg / kg), fucoidan group (50 mg / kg), and combined group. The gallbladder emptying rate was dynamically observed after the contrast agent was perfused through the duodenum.

[0179] Experimental results: At 60 minutes, the gallbladder emptying rate of the combined group was 32%±5%, which was significantly lower than that of the SP-4 group (45%±6%) and the fucoidan group (48%±7%). 2+ The channel (RT-PCR showed that CaV1.2 mRNA was downregulated by 35%) and the M3 receptor antagonism of SP-4 formed a dual regulation, prolonging the cholestasis time to 1.8 times that of a single component.

[0180] Example 12: In vivo nucleation induction experiment of SP-5 modified nano-calcium

[0181] Experimental design:

[0182] Rabbits were induced with bezoar (n=20) and divided into two groups: unmodified nano-calcium group (50 mg / kg) and SP-5 modified nano-calcium group (50 mg / kg, SP-5:CaCO3=1:5). The rabbits were slaughtered and observed after 4 weeks of intervention.

[0183] The number and distribution of gallstones;

[0184] Stone bilirubin calcium content (FTIR quantification);

[0185] Incidence of bile duct obstruction.

[0186] Experimental results:

[0187] The average number of stones in the modified group (8.5±1.3) was greater than that in the unmodified group (4.2±0.9), and the stones were evenly distributed at the gallbladder fundus;

[0188] The bilirubin calcium content reached (68.3±4.1)%, while that of the unmodified group was (42.5±3.7)%, and the bile duct obstruction rate dropped from 25% to 0%, confirming that SP-5 modification can improve the efficiency of crystal nucleus induction and reduce the risk.

[0189] Example 13: Comparative Experiment between Composite Preparation and Traditional Bezoar Inducer

[0190] Experimental design: The cattle bezoar induction experiment (n=30) was divided into 3 groups: a traditional inducer group (taurine + cholesterol), a composite preparation group of the present invention (prepared according to Example 1), and a blank group. The cycle was 4 months.

[0191] Comparison of key indicators:

[0192]

[0193]

[0194] in conclusion:

[0195] The composite preparation achieves a comprehensive breakthrough in the efficiency and safety of bezoar synthesis compared to traditional technologies through the synergistic enhancement of each monomer of sea cucumber peptide (SP-1 / SP-2 improves raw material supply, SP-3 / SP-4 regulates the bile environment, and SP-5 optimizes crystal nucleus formation), combined with the auxiliary effects of fucoidan, kelp polyphenols and nano-calcium.

Claims

1. A preparation for regulating the production of natural bezoar by combining sea cucumber peptide and fucoidan, characterized by: It is composed of sea cucumber peptide components, kelp fucoidan, kelp polyphenols, oyster shell nano-calcium and auxiliary materials; the sea cucumber peptide components contain 5 small molecule peptides, and the amino acid sequences are: SP-1: Ala-Leu-Ser-Pro-Arg-Tyr-Ile-Lys; SP-2: Gly-Pro-Met-Asp-His-Glu-Cys-Trp; SP-3: Val-Ile-Thr-Ala-Gly-Lys-Asn-Pro; SP-4: Ser-Lys-Asp-Glu-Tyr-Pro-Leu-His; SP-5: Cys-Pro-Arg-Gly-Asp-Phe-Tyr-Val.

2. The preparation according to claim 1, characterized in that: The sea cucumber peptide component accounts for 20%-30% of the total mass of the preparation, kelp fucoidan accounts for 30%-40%, kelp polyphenols account for 5%-10%, oyster shell nano calcium accounts for 10%-20%, and the auxiliary materials are microcrystalline cellulose and magnesium stearate 10%-20%.

3. The preparation according to claim 1, characterized in that: The particle size of the oyster shell nano calcium is 50-100 nm, the specific surface area is ≥50 m² / g, and the surface is modified with SP-5 small molecule peptide to form a composite crystal core.

4. The preparation according to claim 1, wherein: The sulfate content of the undaria pinnatifida fucoidan is ≥18%, and the DP value of the brown algae polyphenol in the kelp polyphenol is ≥10.

5. The preparation according to claim 1, characterized in that: The SP-1 enhances bilirubin uptake by binding to the hepatocyte OATP1B1 protein, SP-4 delays bile emptying by inhibiting the gallbladder smooth muscle M3 receptor, and SP-5 modifies the surface of nanocalcium by mimicking the integrin β3 ligand domain.

6. A method for preparing the preparation according to any one of claims 1 to 5, characterized in that: The following steps are involved: SP-1 to SP-5 were prepared by enzymatic hydrolysis-ultrafiltration-chromatographic purification and mixed in equal mass ratios; fucoidan was obtained from kelp through hot water extraction-ethanol precipitation-column chromatography; kelp polyphenols were obtained from kelp residue through ethanol extraction-macroporous resin purification; nano-calcium was prepared by calcination and ball milling of oyster shells and incubated with SP-5 for modification; each component was mixed with excipients to form granules and filled into capsules.

7. The preparation method according to claim 6, wherein: The enzymatic hydrolysis step uses a mixture of neutral protease and trypsin in a ratio of 1:1, and enzymatically hydrolyzes the sea cucumber body wall at 50° C. and pH 7.0 for 4 hours.

8. Use of the preparation according to any one of claims 1 to 5 in the preparation of a bezoar biomimetic synthesis inducer, characterized in that: The preparation achieves efficient synthesis of bezoar by enhancing bilirubin uptake by hepatocytes, delaying gallbladder emptying, inhibiting gallbladder inflammation and promoting crystal nucleus formation, with a bilirubin content of ≥8%.

9. Use of the preparation according to any one of claims 1 to 5 in preparing a functional food for regulating bile metabolism, characterized in that: The preparation is used for improving bile acid metabolism disorder and reducing serum total bile acid level.

10. The sea cucumber peptide SP-5 according to claim 1, characterized in that: The amino acid sequence is Cys-Pro-Arg-Gly-Asp-Phe-Tyr-Val, which is used to modify the surface of oyster shell nanocalcium and promote the deposition of bilirubin calcium salt.

Images