Anti-antler plate oligopeptide as well as preparation method and application thereof

Abstract

The invention discloses an antler plate oligopeptide as well as a preparation method and application thereof, and relates to the technical field of bioactive peptides. The preparation method comprises the following steps: extracting an antler plate protein crude extract by taking antler plates as a raw material, preparing short peptides with antler plate molecular weight of less than 3k through pepsin and trypsin hydrolysis, and performing Sephadex G-15 gel chromatography to obtain the antler plate short peptides. The prepared antler plate oligopeptide is low in molecular weight and high in hydrophobicity, easily penetrates through a biological membrane barrier to reach an in-vivo effective part, fully plays the biological activity effect of small molecule peptide, can effectively promote C28 / I2 cartilage cell proliferation, can increase the expression of osteoarthritis related phenotype protein Collagen II in an in-vitro model, can reduce the expression of MMP3, MMP13 and ADAMTS-5, and can be used for treating osteoarthritis. Meanwhile, cartilage matrix degradation in an osteoarthritis rat model is inhibited, cartilage injury is improved, and inflammatory response induced by osteoarthritis is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of bioactive peptides, and particularly relates to a short peptide from deer antler disc, a preparation method thereof, and an application thereof. Background Art

[0002] Deer antler disc, commonly known as deer antler plate, deer flower plate and deer antler cap, is the ossified horn of Cervus elaphus Linnaeus or Cervus Nippon Temminck of the deer family, or the horn base shed in the following spring after sawing the antler. According to the records in the Pharmacopoeia of the People's Republic of China (2020 Edition), deer antler disc is warm in nature, salty in taste, and belongs to the kidney and liver meridians. It has the effects of warming the kidney yang, strengthening tendons and bones, promoting blood circulation and reducing swelling. Deer antler disc is an animal Chinese medicinal material rich in various components and pharmacological activities. Its components mainly include proteins, polypeptides, amino acids, cholesterol, lipids, inorganic substances and minerals, etc. Among them, water-soluble proteins and peptide substances are its main active components, which not only have relatively high contents, but also have rich medicinal values. Modern pharmacological studies have shown that deer antler disc has pharmacological effects such as anti-inflammatory, antioxidant, delaying cartilage damage, treating hyperplasia of mammary glands, regulating immunity, and antibacterial.

[0003] Osteoarthritis is a chronic degenerative joint disease characterized by damage to joint tissues. In the initial stage of osteoarthritis, it may be triggered by injuries or pathogenic factors of cartilage, ligaments or other joint tissues, and then lead to synovial inflammation, oxidative stress, chondrocyte apoptosis, degradation of the extracellular matrix of chondrocytes, subchondral bone sclerosis and osteophyte formation, etc. The specific manifestations are joint stiffness, deformity, pain and decline of joint function. According to traditional Chinese medicine theory, the root cause of knee osteoarthritis is the deficiency of the liver, spleen and kidney and the imbalance of tendons and bones. Its pathogenesis is the deficiency of the liver and kidney, qi deficiency and blood stasis, spleen deficiency and failure of transportation, external pathogenic invasion, and trauma and strain.

[0004] In the past few decades, people have mostly only focused on the nutritional components and medicinal values of deer by-products such as deer antler and deer penis, and ignored deer antler disc, an animal Chinese medicinal material with broad market prospects. Deer antler disc is the only completely renewable organ of mammals, which can be obtained without harming the animal. Its rich biological activities and the advantages of resource recycling are worthy of extensive attention from researchers. In recent years, some scholars have isolated bioactive medicinal components from deer antler disc and verified their different effects through pharmacological experiments. There are also compound traditional Chinese medicines such as Yougui Pills and Xitongkang, which use deer antler as the monarch drug, for treating knee osteoarthritis. As one of the main active components of deer antler disc, polypeptides are inseparable from the pharmacological effects it exerts.

[0005] Bioactive peptides are peptide segments with biological functions composed of 2 - 20 amino acids, formed by different arrangements and combinations. The physiological activity of active peptides is closely related to the types and sequences of their amino acid compositions, including amino acid composition, types of terminal amino acid residues, peptide chain length, peptide surface charge, and hydrophilic and hydrophobic characteristics. Different amino acid sequences and types can possess different physiological activities, such as participating in or regulating the human immune system, endocrine system, cardiovascular system, and nervous system, and affecting the utilization rate of nutrients by the body. In addition, low-molecular-weight active peptides will produce shorter active peptide fragments due to hydrolysis, thus fully exposing the active amino acid residue sites. Such peptides are more conducive to interacting with other substances, crossing the biological membrane barrier, and reaching the effective parts in the body. Summary of the Invention

[0006] The object of the present invention is to provide a short peptide from deer antler pedicle, which can effectively promote the proliferation of C28 / I2 chondrocytes, and can increase the expression of osteoarthritis-related phenotypic protein Collagen II in an in vitro model, and reduce the expression of MMP3, MMP13, and ADAMTS-5.

[0007] Another object of the present invention is to provide a preparation method for the above-mentioned short peptide from deer antler pedicle.

[0008] The third object of the present invention is to provide the application of the above-mentioned short peptide from deer antler pedicle in the preparation of products for promoting chondrocyte proliferation and improving the expression of osteoarthritis-related phenotypic proteins in chondrocytes.

[0009] The third object of the present invention is to provide the application of the above-mentioned short peptide from deer antler pedicle in the preparation of products for delaying cartilage damage in rats with osteoarthritis and reducing the inflammatory response.

[0010] The object of the present invention is achieved by the following technical solution: A short peptide from deer antler pedicle, comprising 10 peptide segments, with the molecular mass range of the peptide segments being 412.4 - 862.5 Da, and the sequence length range being 4 - 10 amino acids.

[0011] Further, the amino acid sequence of the short peptide is Leu-Leu-Gly-Asp-Val (LLGDV), Phe-Gly-Tyr-

[0012] Asp-Gly-Asp-Phe (FGYDGDF), Phe-Ser-Gly-Leu-Asp-Gln (FSGLDQ), Phe-Gly-Phe-

[0013] Asp-Gly-Asp-Phe (FGFDGDF), Val-Gly-Pro-Val (VGPV), Phe-Gly-Phe-Asp-Gly-Asp

[0014] (FGFDGD), Val-Gly-Pro-Glu-Pro-Gln (VGPEPQ), Ala-Ala-Gly-Pro-Ala-Gly-Pro-

[0015] Leu-Gly-Ser (AAGPAGPLGS), Ala-Gly-Pro-Ala-Gly-Ala (AGPAGA), Val-Gly-Pro-

[0016] Val-Gly-Pro-Ala-Gly-Pro-Leu (VGPVGPAGPL).

[0017] The preparation method of the above-mentioned antler pedicle short peptides uses antler pedicle as the raw material to extract the crude antler pedicle protein, prepares short peptides with a molecular weight below 3k of antler pedicle by hydrolysis with pepsin and trypsin, and obtains antler pedicle short peptides through Sephadex G-15 gel chromatography.

[0018] Furthermore, for the extraction of the crude protein extract, antler pedicle powder is added to NaOH solution, heated and magnetically stirred, centrifuged, the supernatant is retained, the precipitate is taken and extracted once more, and the two supernatants are combined to obtain an aqueous extract; (NH4)2SO4 is gradually and slowly added to the aqueous extract to a saturation of 80%, this solution is placed in an environment at 4°C for 10 h to obtain a protein suspension, centrifuged again, the precipitate is taken, 0.05M Tris-HCl buffer solution (pH 7.25) is added until the precipitate is completely dissolved, desalted by dialysis, and freeze-dried to obtain the crude antler pedicle protein CNCP.

[0019] Furthermore, for the hydrolysis, the crude antler pedicle protein CNCP is used as the substrate, dissolved in 0.01M Tris-HCl to 12.5 mg / mL, pepsin is added and stirred for 3 h, then trypsin is added and stirred for 3 h, followed by freeze-drying, distilled water is added to a final concentration of 10 mg / mL, centrifuged, and the permeate is short peptides with a molecular weight below 3k, named CNCPs. The addition amount of pepsin is 7000 u / g and pH = 2.9, and the addition amount of trypsin is 7000 u / g and pH = 7.9.

[0020] Furthermore, for the Sephadex G-15 gel chromatography, the CNCPs powder is fully dissolved in ultrapure water to a final concentration of 12.5 mg / mL, the sample loading solution is slowly added along the column wall to the Sephadex G-15 gel chromatography column, and eluted with ultrapure water at a flow rate of 0.3 mL / min.

[0021] The application of the above-mentioned antler pedicle short peptides is in the preparation of products for promoting chondrocyte proliferation and improving the expression of osteoarthritis-related phenotypic proteins in chondrocytes.

[0022] The application of the above-mentioned antler pedicle short peptide in the preparation of a product for delaying cartilage damage in rats with osteoarthritis and reducing the inflammatory response.

[0023] The present invention has the following technical effects:

[0024] The antler pedicle short peptide prepared by the present invention has a low molecular weight, strong hydrophobicity, is easy to penetrate the biological membrane barrier, reaches the effective part in the body, gives full play to the biological activity of small molecule peptides, can effectively promote the proliferation of C28 / I2 chondrocytes, and can increase the expression of osteoarthritis-related phenotypic protein Collagen II in the in vitro model, reduce the expression of MMP3, MMP13, and ADAMTS-5, and at the same time inhibit the degradation of cartilage matrix in the osteoarthritis rat model, improve cartilage damage, and reduce the inflammatory response induced by osteoarthritis.

[0025] The extraction method of the present application by combining alkali extraction with ammonium sulfate precipitation can maximize the extraction rate of substrate proteins, and the combined use of pepsin and trypsin can hydrolyze the drug, simulate the reaction of the drug in the human gastrointestinal tract, improve the bioavailability of the drug and the release of small molecule bioactive peptide segments. The ultrafiltration molecular sieve can directly separate the short peptide and improve the separation efficiency. Description of the Drawings

[0026] Figure 1 It is the elution curve of the separation and purification of Sephadex G-15 of the present invention;

[0027] Figure 2 It is the influence diagram of CNCPs-I-IV of the present invention on the proliferation of C28 / I2 cells;

[0028] Figure 3 It is the influence of CNCPs-I-IV of the present invention on osteoarthritis-related phenotypic proteins in chondrocytes;

[0029] Figure 4 It is the LC-MS / MS identification mass spectrum diagram of CNCPs-III of the present invention;

[0030] Figure 5 It is the pathological section diagram of safranin O-fast green staining of cartilage of CNCPs-III of the present invention on osteoarthritis model rats

[0031] Figure 6 It is the influence of CNCPs-III of the present invention on inflammatory factors in the serum of osteoarthritis model rats. Detailed Embodiments

[0032] The present invention separates and purifies a short peptide from deer antler pedicles through steps such as salting out, enzymatic hydrolysis, ultrafiltration molecular cut-off, and gel filtration chromatography (Sephadex G-15) column chromatography, and identifies its structural sequence by LC-MS / MS. The pharmacodynamics of the deer antler pedicle short peptide in delaying osteoarthritis in vitro and in vivo is evaluated, laying an experimental foundation and theoretical basis for the development and clinical application of the deer antler pedicle short peptide.

[0033] The present invention will be described in detail below in conjunction with the embodiments:

[0034] Example 1

[0035] The extraction, separation, and purification of a deer antler pedicle short peptide specifically include the following steps:

[0036] Weigh 200 g of deer antler pedicle powder, add 5 L of NaOH solution with a pH value of 9.6, heat to 52 °C, stir magnetically for 3 h, centrifuge at 5000 rmp at room temperature for 30 min, retain the supernatant, take the precipitate and extract it once more, then combine the two supernatants to obtain an aqueous extract; gradually and slowly add (NH4)2SO4 to the aqueous extract to make its saturation reach 80%, place this solution in an environment at 4 °C for 10 h to obtain a protein suspension, centrifuge at 5000 rmp at room temperature for 30 min, take the precipitate, add 0.05 M Tris-HCl buffer solution (pH 7.25) until the precipitate is completely dissolved, dialyze to remove salts, and freeze-dry to obtain a crude extract of deer antler pedicle protein CNCP;

[0037] Dissolve the CNCP powder with 0.01 M Tris-HCl to 12.5 mg / mL, place it at 37 °C, first add pepsin (7000 u / g, pH = 2.9) and stir for 3 h, then add trypsin (7000 u / g, pH = 7.9) and stir for 3 h, and then freeze-dry. Take the dried powder and add distilled water to a final concentration of 10 mg / mL, centrifuge in an ultrafiltration centrifuge tube (3k), and the permeate is a short peptide with a molecular weight below 3k, named CNCPs.

[0038] Take an appropriate amount of CNCPs powder and fully dissolve it with ultrapure water to a final concentration of 12.5 mg / mL. Slowly add the sample solution along the column wall into a Sephadex G-15 gel chromatography column, elute with ultrapure water at a flow rate of 0.3 mL / min, use an automatic partial sampler to collect the eluate, 1.5 mL per tube, and collect 100 tubes; measure the absorbance at a wavelength of 220 nm every other tube, plot the Sephadex G-15 elution curve with the number of tubes detected as the abscissa and the absorbance value at 220 nm as the ordinate. The results are shown in Figure 1, in which four components, namely CNCPs-I, CNCPs-II, CNCPs-III, and CNCPs-IV, were isolated. After detecting the activity of improving osteoarthritis in vitro, the CNCPs-III with the best activity was used for subsequent experiments.

[0039] Example 2

[0040] Activity determination for improving the injury of immortalized human chondrocyte cell line C28 / I2

[0041] C28 / I2 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and placed in an incubator at 37°C and 5% CO2. The cells were treated with 10 ng / mL IL-1β in PBS to establish an in vitro model of OA cartilage injury. DMSO was used to dissolve the drugs CNCPs-I to IV.

[0042] 1) Cell proliferation

[0043] The cells were seeded into 96-well plates for culture and stimulated with sera containing different doses of CNCPs-I to IV for 24 hours. Then, 10 μL of CCK-8 reagent was added to each well and incubated at 37°C for 4 hours. The optical density value was measured at a wavelength of 450 nm. The experimental results showed that when the concentrations of CNCPs-I to IV were 0 - 100 μg / mL, there was no obvious toxicity to C28 / I2 cells. Therefore, in subsequent experiments, a concentration of 0 - 100 μg / mL could be selected to investigate the effect of CNCPs-I to IV on the viability of IL-1β-induced C28 / I2 chondrocyte injury. As Figure 2 , when the concentrations of CNCPs-I to IV were 100 μg / mL, the effect on the viability of IL-1β-induced chondrocyte injury was the most significant. Therefore, a concentration of 100 μg / mL was selected for the in vitro experiment. When the concentration of each group was 100 μg / mL, among them, CNCPs-III had the most significant effect on the proliferation of chondrocyte injury cells ( Figure 3 A).

[0044] 2) Effects on the protein and mRNA expression of articular cartilage-related phenotypic proteins (Collagen II, MMP3, MMP13, ADAMTS-5)

[0045] To screen for the best pharmacodynamic components of CNCPs-I to IV in delaying cartilage damage, the expression of osteoarthritis-related phenotypic proteins was detected by Western blot and qRT-PCR methods. The results showed that compared with the IL-1β treatment group, the expression levels of Collagen II mRNA and protein in the CNCPs-I to IV groups were relatively increased, while the expression levels of MMP3, MMP13, and ADAMTS-5 were significantly decreased. Among them, the effect of CNCPs-III was the most significant (p<0.01)( Figure 3 B-J). In the OA pathological environment, ADAMTS and MMPs are considered to be the most important proteases involved in the degradation of the chondrocyte extracellular matrix. The extracellular matrix is mainly composed of Collagen II and ACAN, which are the main components of cartilage. It can enable articular cartilage to adapt to biomechanical stress during joint movement and plays an important role in maintaining knee joint homeostasis.

[0046] The above results indicate that in the in vitro OA model, CNCPs-I to IV all have an impact on OA-related phenotypic proteins. They inhibit the degradation of the chondrocyte extracellular matrix and reduce the expression of proteases such as ADAMTS and MMPs. Among them, CNCPs-III has the best effect in reducing protease expression and delaying cartilage damage. Therefore, the CNCPs-III fraction was selected for subsequent structure identification.

[0047] Example 3

[0048] Structure identification of CNCPs-III

[0049] LC-MS / MS was used to identify the peptide sequences in CNCPs-III with the best OA-improving activity. The peptide sequences in the raw mass spectrometry files were analyzed by the PEAKS de novo method. A total of 10 main peptide segments were identified. The molecular mass range of the peptide segments was 412.4 - 862.5 Da, and the sequence length range was 4 - 10 amino acids. Peptide segments with a large number of hydrophobic amino acid residues (Ala, Gly, Val, Leu, Pro, Phe) in the amino acid sequence. Such as Figure 4, the amino acid sequences are Leu-Leu-Gly-Asp-Val (LLGDV), Phe-Gly-Tyr-Asp-Gly-Asp-Phe (FGYDGDF), Phe-Ser-Gly-Leu-Asp-Gln (FSGLDQ), Phe-Gly-Phe-Asp-Gly-Asp-Phe (FGFDGDF), Val-Gly-Pro-Val (VGPV), Phe-Gly-Phe-Asp-Gly-Asp (FGFDGD), Val-Gly-Pro-Glu-Pro-Gln (VGPEPQ), Ala-Ala-Gly-Pro-Ala-Gly-Pro-Leu-Gly-Ser (AAGPAGPLGS), Ala-Gly-Pro-Ala-Gly-Ala (AGPAGA), Val-Gly-Pro-Val-Gly-Pro-Ala-Gly-Pro-Leu (VGPVGPAGPL). The peptide sequence information is shown in Table 1.

[0050] Table 1 Peptide Sequence Information

[0051]

[0052] Example 4

[0053] Pharmacodynamic Evaluation of Improving Osteoarthritis in Rats

[0054] Twenty-four male SD rats at 6 weeks of age with a body weight of 180 - 220 g were selected. After 7 days of adaptive feeding, the rats were divided into 3 groups by the random number table method, namely the blank group (Con), the model group (Mod), and the CNCPs-III group (n = 8). Except for the rats in the Con group that were injected with normal saline into the joint cavity, on the 1st, 4th, and 7th days of the experiment, the hind leg joint cavities of the rats were injected with 4% papain - 0.03 mol / L cysteine to induce an osteoarthritis rat model. From the 21st day, the rats in the Con group and the Mod group were intragastrically administered distilled water, and the rats in the CNCPs-III group were intragastrically administered 40 mg / kg of CNCPs-III for 30 days. After the experiment was terminated, the rats were intraperitoneally injected with 150 mg / kg of sodium pentobarbital. Abdominal aortic blood samples were taken for enzyme-linked immunosorbent assay, and joint tissues were taken out for making pathological sections.

[0055] 1) Pathological Analysis of Cartilage Tissue in Osteoarthritis Rats

[0056] The joint tissues were fixed in 4% paraformaldehyde for 48 hours and decalcified in 10% EDTA for 30 days, then embedded and sectioned along the sagittal plane with a thickness of 4 μm. After the sections were stained with safranin O and fast green, pathological evaluation was performed under an optical microscope. The results are as Figure 5As shown, safranin-fast green staining of cartilage tissue sections in the Con group showed that the cartilage matrix was brightly red-stained, while the subchondral bone in the superficial layer was green. In the sections of the Mod group, it was visible that the safranin staining was significantly weakened, indicating a reduced thickness of the cartilage surface layer, erosion of the superficial cartilage, and severe degradation of the cartilage matrix. After administration of CNCPs-III, strong positive staining was presented, indicating that the degradation of proteoglycans and cartilage erosion were improved.

[0057] 2) Enzyme-linked immunosorbent assay for the levels of inflammatory factors in rat serum

[0058] As Figure 6 , it shows the effects of CNCPs-III on the contents of IL-1β, IL-18, and TNF-α inflammatory factors in the serum of osteoarthritis model rats. Compared with the Con group, the contents of IL-1β, IL-18, and TNF-α in the Mod group were significantly increased (p < 0.001), indicating that the pathogenesis of osteoarthritis is related to the inflammatory response. Compared with the Mod group, CNCPs-III could significantly reduce the contents of IL-1β, IL-18, and TNF-α in the serum of OA rats (P < 0.001). These results indicate that CNCPs-III can regulate the levels of inflammation-related factors in osteoarthritis.

[0059] In the inflammatory response of joints, IL-1β is considered a key pro-inflammatory factor. In addition, various pro-inflammatory cytokines such as IL-6, IL-18, and TNF-α are highly expressed in OA cartilage, which can significantly promote the expression of matrix metalloproteinases MMPs and ADAMTS, inhibit the synthesis of ECM, and induce apoptosis, thus accelerating cartilage degeneration, promoting extracellular matrix degradation, and joint dysfunction. The experimental results show that CNCPs-III can significantly reduce the levels of inflammatory factors in rat serum and improve the inflammatory response caused by osteoarthritis.

Claims

1. A short peptide of deer antler pedicle, characterized in that, It contains 10 peptide segments with a molecular mass range of 412.4 - 862.5 Da and a sequence length range of 4 - 10 amino acids.

2. The antler pedestal short peptide according to claim 1, characterized in that, The amino acid sequences of the short peptides are Leu-Leu-Gly-Asp-Val (LLGDV), Phe-Gly-Tyr-Asp-Gly-Asp-Phe (FGYDGDF), Phe-Ser-Gly-Leu-Asp-Gln (FSGLDQ), Phe-Gly-Phe-Asp-Gly-Asp-Phe (FGFDGDF), Val-Gly-Pro-Val (VGPV), Phe-Gly-Phe-Asp-Gly-Asp (FGFDGD), Val-Gly-Pro-Glu-Pro-Gln (VGPEPQ), Ala-Ala-Gly-Pro-Ala-Gly-Pro-Leu-Gly-Ser (AAGPAGPLGS), Ala-Gly-Pro-Ala-Gly-Ala (AGPAGA), Val-Gly-Pro-Val-Gly-Pro-Ala-Gly-Pro-Leu (VGPVGPAGPL).

3. A method for preparing the antler disk short peptide according to any one of claims 1-2, characterized in that: Taking antler pedicle as the raw material, the crude extract of antler pedicle protein is extracted. Short peptides with a molecular weight below 3k are prepared by hydrolysis with pepsin and trypsin, and the short peptides of antler pedicle are obtained through Sephadex G-15 gel chromatography.

4. A preparation method of the antler disk short peptide as described in claim 3, characterized in that: The crude protein extract is obtained by adding antler pedicle powder to NaOH solution, heating and stirring magnetically, followed by centrifugation. The supernatant is retained, and the precipitate is extracted one more time. Then the two supernatants are combined to obtain an aqueous extract. (NH4)2SO4 is gradually and slowly added to the aqueous extract to reach a saturation of 80%. This solution is placed in a 4°C environment for 10 h to obtain a protein suspension. After centrifugation again, the precipitate is taken, and 0.05M Tris-HCl buffer solution (pH 7.25) is added until the precipitate is completely dissolved. After dialysis to remove salts and freeze-drying, the crude extract of antler pedicle protein CNCP is obtained.

5. A preparation method of the antler pedicle short peptide as described in claim 4, characterized in that: For the hydrolysis, the crude extract of antler pedicle protein CNCP is used as the substrate and dissolved in 0.01M Tris-HCl to a concentration of 12.5 mg / mL. Pepsin is added and stirred for 3 h, then trypsin is added and stirred for 3 h, followed by freeze-drying. Distilled water is added to a final concentration of 10 mg / mL, and after centrifugation, the permeate is the short peptide with a molecular weight below 3k, named CNCPs. The addition amount of pepsin is 7000 u / g and the pH is 2.9, and the addition amount of trypsin is 7000 u / g and the pH is 7.

9.

6. A preparation method of the antler disc short peptide as described in claim 4, characterized in that: For the Sephadex G-15 gel chromatography, the CNCPs powder is fully dissolved in ultrapure water to a final concentration of 12.5 mg / mL. The sample loading solution is slowly added along the column wall into the Sephadex G-15 gel chromatography column, and eluted with ultrapure water at a flow rate of 0.3 mL / min.

7. The application of the antler pedicle short peptide according to claim 1, wherein: Use in the preparation of a product for promoting chondrocyte proliferation and improving the expression of osteoarthritis-related phenotypic proteins in chondrocytes.

8. Use of the antler disk short peptide according to claim 1, characterized in that: Use in the preparation of a product for delaying cartilage damage in rats with osteoarthritis and reducing the inflammatory response.

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