Active peptides, active peptide compositions and use thereof in the manufacture of products having anti-inflammatory effects

Abstract

The present application relates to the field of biomedical technology, and specifically discloses an active peptide, an active peptide composition and application of the active peptide and the active peptide composition in preparation of products with anti-inflammatory effect. The amino acid sequence of the active peptide is shown in SEQ ID No. 1 or SEQ ID No. 2. The active peptide composition comprises the active peptide with the amino acid sequence shown in SEQ ID No. 1 and SEQ ID No. 2. Research shows that the active peptide and the active peptide composition have anti-inflammatory activity; in particular, have excellent anti-inflammatory or inflammatory damage effect on intestinal epithelial cells and can improve the activity of intestinal epithelial cells; therefore, the active peptide and the active peptide composition are used as effective components to prepare food or medicine with anti-inflammatory effect, in particular, food or medicine with anti-inflammatory or inflammatory damage effect on intestinal epithelial cells and can improve the activity of intestinal epithelial cells, which has important application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an active peptide, an active peptide composition, and its application in the preparation of products with anti-inflammatory effects. Background Technology

[0002] Chronic colitis is characterized by its recurrent and frequent occurrence, with multiple causes. Because it easily leads to inflammatory edema, ulceration, and bleeding lesions in the intestines, it is also known as ulcerative colitis. Chronic colitis is concentrated in the sigmoid colon, rectum, and even the entire colon. The main symptoms include abnormal bowel movements, abdominal pain, bloody stools, and abnormal anal sensations. Currently, treatment for chronic colitis mainly includes traditional Chinese medicine and Western medicine. Traditional Chinese medicine focuses on strengthening the spleen and kidneys, replenishing qi and eliminating dampness, clearing heat and detoxifying, and promoting blood circulation and removing blood stasis, aiming to treat both the symptoms and the root cause. Western medicine mainly uses hormones, immunosuppressants, and biological agents.

[0003] Probiotics can colonize the human body and promote nutrient absorption and maintain intestinal health by regulating the balance of intestinal flora. They are safe, non-pathogenic, and tolerant of gastric acid and bile salts, making them a type of live microorganism beneficial to the host. Studies have shown that changes in the intestinal microecology are closely related to the development of chronic inflammatory diseases both in and outside the intestine, and probiotics can play a protective role for patients with inflammatory bowel disease, demonstrating great potential and advantages in its treatment.

[0004] Oral biotherapy products and engineered bacteria have provided new and broad treatment platforms for probiotics in treating inflammatory bowel disease. Probiotics or their secreted short-chain fatty acids play an important role in the treatment of intestinal-related diseases; short-chain fatty acids produced by the gut microbiota can directly affect cell metabolism and regulate intestinal immune responses. Currently, however, there are few reports on whether oligopeptides produced after probiotic secretion or lysis possess anti-inflammatory activity and whether they can be used to treat inflammatory bowel disease. Summary of the Invention

[0005] To overcome at least one of the technical problems existing in the prior art, the present invention first provides an active peptide and an active peptide composition. Studies have shown that the active peptide and the active peptide composition have anti-inflammatory activity; in particular, they have excellent anti-inflammatory or anti-inflammatory damage to intestinal epithelial cells and enhance the activity of intestinal epithelial cells.

[0006] The above-mentioned technical problem to be solved by the present invention is achieved through the following technical solution:

[0007] An active peptide, wherein the amino acid sequence of the active peptide is shown in SEQ ID No. 1 or SEQ ID No. 2.

[0008] The amino acid sequence shown in SEQ ID No. 1 is Gly-Glu-His-Pro-Arg (hereinafter abbreviated as LOP-a).

[0009] The amino acid sequence shown in SEQ ID No. 2 is His-Lys-Arg-Lys-Glu (hereinafter abbreviated as LOP-b).

[0010] The active peptides with the amino acid sequences shown in SEQ ID No. 1 and SEQ ID No. 2 can be isolated from Lactobacillus rhamnosus or synthesized by conventional peptide synthesis methods.

[0011] The present invention also provides an active peptide composition comprising SEQ ID No. 1

[0012] The active peptide with the amino acid sequence shown in SEQ ID No. 2.

[0013] The inventors were surprised to discover in further research that the images shown in SEQ ID No. 1 and SEQ ID No. 2...

[0014] The anti-inflammatory activity of the combination of active peptides with amino acid sequences, especially the anti-inflammatory or inflammatory damage effect on intestinal epithelial cells and the effect of improving the activity of intestinal epithelial cells, is further superior to that of the active peptides with amino acid sequences shown in SEQ ID No. 1 or SEQ ID No. 2 alone; the combination of the two has a synergistic effect.

[0015] Preferably, the mass ratio of the active peptides of the amino acid sequences shown in SEQ ID No. 1 and SEQ ID No. 2 is 1:1-10.

[0016] More preferably, the mass ratio of the active peptides of the amino acid sequences shown in SEQ ID No. 1 and SEQ ID No. 2 is 1:1-5.

[0017] Most preferably, the mass ratio of the active peptides of the amino acid sequences shown in SEQ ID No. 1 and SEQ ID No. 2 is 1:1.

[0018] The present invention also provides the use of the above-mentioned active peptides or active peptide compositions in the preparation of products with anti-inflammatory effects.

[0019] Preferably, the anti-inflammatory effect refers to the anti-inflammatory effect on intestinal epithelial cell inflammation or inflammatory damage.

[0020] The present invention also provides the use of the above-mentioned active peptides or active peptide compositions in the preparation of products that enhance the activity of intestinal cells.

[0021] Preferably, the intestine is the colon.

[0022] Preferably, the product is a pharmaceutical product.

[0023] Preferably, the drug uses the above-mentioned active peptide or active peptide composition as the active ingredient and further includes pharmaceutical excipients.

[0024] Preferably, in the pharmaceutical product, the active peptide or active peptide composition has a mass percentage of 1% to 90%, 5% to 50%, or 10% to 50%.

[0025] Beneficial Effects: This invention provides a novel bioactive peptide and bioactive peptide composition. Studies have shown that the bioactive peptide and bioactive peptide composition possess anti-inflammatory activity, particularly exhibiting excellent anti-inflammatory or anti-inflammatory damage effects on intestinal epithelial cells and enhancing the activity of intestinal epithelial cells. Therefore, using the bioactive peptide and bioactive peptide composition of this invention as an active ingredient in the preparation of pharmaceuticals with anti-inflammatory effects, especially those with anti-inflammatory or anti-inflammatory damage effects on intestinal epithelial cells and enhanced intestinal epithelial cell activity, has significant application prospects. Furthermore, the probiotic bioactive oligopeptides of this invention can be isolated and purified from *Lactobacillus rhamnosus* or obtained through synthetic methods. The preparation process is simple and convenient, enabling large-scale production and application. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only drawings of some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 This is the MS plot of LOP-a.

[0028] Figure 2 This is the HPLC chromatogram of LOP-a.

[0029] Figure 3 This is the MS plot of LOP-b.

[0030] Figure 4 This is the HPLC chromatogram of LOP-b.

[0031] Figure 5 The effects of LOP-a, LOP-b, and LOP-t on the viability of intestinal epithelial cells.

[0032] Figure 6 The effects of LOP-a, LOP-b, and LOP-t on ROS, MDA content, and SOD activity in intestinal epithelial cells were investigated.

[0033] Figure 7 The effects of LOP-a, LOP-b, and LOP-t on the secretion of inflammatory cytokines in intestinal epithelial cells were investigated.

[0034] Figure 8 The effects of LOP-a, LOP-b, and LOP-t on the expression of proteins related to the NF-κB and NLRP3 inflammasome signaling pathway in intestinal epithelial cells were investigated.

[0035] Figure 9 The effects of LOP-a, LOP-b, and LOP-t on the expression of tight junction proteins in intestinal epithelial cells were investigated. Detailed Implementation

[0036] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] The LOP-a and LOP-b in the following embodiments were synthesized using conventional methods in the art. The instruments, equipment and condition parameters not disclosed in detail in the synthesis steps are all well-known technologies to those skilled in the art.

[0038] Example 1: Artificial Synthesis Method of LOP-a

[0039] 100 mg of Fmoc-Gly Wang Resin was placed in a solid-phase synthesis tube, N,N-dimethylformamide (DMF) was added, and the mixture was allowed to stand for 30 minutes to allow the resin to fully swell. The solvent was filtered off, and then piperidine DMF solution was added. After shaking, the solvent was filtered off. Fmoc-Glu-OH, 1-hydroxybenzotriazole, and O-benzotriazole-tetramethylurea hexafluorophosphate were dissolved in DMF. After adding N,N-diisopropylethylamine and mixing well, the mixture was activated in the dark and added to the resin. The mixture was stirred under nitrogen at 25°C for 2 hours. After filtration, the resin was washed with DMF and dichloromethane sequentially, and the solvent was dried under vacuum. The above steps were repeated, and the activated Fmoc-His-OH, Fmoc-Pro-OH, and Fmoc-Arg-OH were added to the resin sequentially. The mixture was stirred under nitrogen at 25°C. After the reaction was complete, the resin was washed, and the filtrate was rotary evaporated to obtain a precipitate. After freeze-drying, LOP-a was obtained. The amino acid sequence of LOP-a is Gly-Glu-His-Pro-Arg (i.e., the amino acid sequence shown in SEQ ID No. 1).

[0040] Example 2: Artificial Synthesis Method of LOP-b

[0041] 100 mg of Fmoc-His Wang Resin was placed in a solid-phase synthesis tube, N,N-dimethylformamide (DMF) was added, and the mixture was allowed to stand for 30 minutes to allow the resin to fully swell. The solvent was filtered off, and then piperidine DMF solution was added. After shaking, the solvent was filtered off. Fmoc-Lys-OH, 1-hydroxybenzotriazole, and O-benzotriazole-tetramethylurea hexafluorophosphate were dissolved in DMF. After adding N,N-diisopropylethylamine and mixing well, the mixture was activated in the dark and added to the resin. The mixture was stirred under nitrogen at 25°C for 2 hours. After filtration, the resin was washed successively with DMF and dichloromethane, and the solvent was dried under vacuum. The above steps were repeated, and the activated Fmoc-Arg-OH, Fmoc-Lys-OH, and Fmoc-Glu-OH were added to the resin successively. The mixture was stirred under nitrogen at 25°C. After the reaction was complete, the resin was washed, and the filtrate was rotary evaporated to obtain a precipitate. After freeze-drying, LOP-b was obtained. The amino acid sequence of LOP-b is His-Lys-Arg-Lys-Glu (i.e., the amino acid sequence shown in SEQ ID No. 2).

[0042] The structural analysis and molecular weight determination of LOP-a and LOP-b were also performed using electrospray ionization mass spectrometry (HPLC-ESI-MS) on a Q-TOF mass spectrometer. The mass range was set to m / z 50-1500. Mass spectrometry data were acquired in positive mode under the following conditions: CAD gas flow rate, 7 L / min; dry gas temperature, 550°C; ion spray voltage, 5500 V; and deaggregation potential, 80 V.

[0043] Finally, the amino acid sequences of LOP-a and LOP-b were determined based on mass spectrometry data and the Edman degradation method, as shown in SEQ ID No. 1 and SEQ ID No. 2.

[0044] Example 3: Active peptide composition

[0045] The active peptide composition is obtained by mixing LOP-a and LOP-b in a mass ratio of 1:1 until homogeneous.

[0046] Example 4: Active peptide composition

[0047] The active peptide composition is obtained by mixing LOP-a and LOP-b in a mass ratio of 1:10 until homogeneous.

[0048] Example 5: Active peptide composition

[0049] The active peptide composition is obtained by mixing LOP-a and LOP-b in a mass ratio of 1:5 until homogeneous.

[0050] Effect Example

[0051] The experimental subjects in the following effect examples are LOP-a, LOP-b, and the active peptide composition (abbreviated as LOP-t) formed by mixing LOP-a and LOP-b in a mass ratio of 1:1 in Example 3.

[0052] HCoEpiC (human normal colonic epithelial cells) at 5×10 3 Hcoepic cells were seeded at a density of 1 mg / mL in Costa 96-well plates and incubated with 1% DSS for 12 hours. Then, the cells were washed twice with cold PBS and cultured for 24 hours with LOP-a (1 mg / mL), LOP-b (1 mg / mL), LOP-t (1 mg / mL), and Lactobacillus rhamnosus sonicated lysate (LLW, 1 mg / mL). Cell viability was assessed using the CCK 8 assay.

[0053] 5×10 4 Hcoepic cells were incubated in 6-well plates with 1% DSS for 12 hours. Then, the Hcoepic cells were washed twice with cold PBS and incubated with LOP-a (1 mg / mL), LOP-b (1 mg / mL), and LOP-t (1 mg / mL) for 24 hours, respectively. The supernatant from the cell culture was collected. The levels of inflammatory epithelial cytokines were measured using an ELISA kit. Cells were collected, proteins were extracted, and their effects on related signaling pathways were investigated using Western blotting.

[0054] The experimental results are as follows:

[0055] The experimental results (Figure 5A) showed that after co-culturing Hcoepic cells with LOP-a (1 mg / mL), LOP-b (1 mg / mL), LOP-t (1 mg / mL), and Lactobacillus rhamnosus ultrasonic lysate (LLW, 1 mg / mL) for 24 hours, the viability of Hcoepic cells was significantly stronger than that of the blank control group (cell viability of the blank group was set to 100%), indicating that LOP-a, LOP-b, LOP-t, and LLW have a strong promoting effect on the cell viability of cultured normal human colonic epithelial cells in vitro. Compared with the blank control group, LOP-a, LOP-b, LOP-t, and LLW increased cell viability by 11.3% (p<0.05), 13.9% (p<0.05), 20.1% (p<0.01), and 7.34% (p<0.05), respectively. Meanwhile, the active peptide composition LOP-t is superior to LOP-a, LOP-b, or Lactobacillus rhamnosus lysate in promoting the activity of normal human colonic epithelial cells at the same concentration.

[0056] The experimental results (Figure 5B) showed that DSS exposure significantly reduced Hcoepic cell viability. We hypothesize that DSS exposure damages Hcoepic cells, thereby reducing their viability. LOP-a, LOP-b, and LOP-t treatments significantly inhibited DSS-induced reduction in intestinal epithelial cell viability. Compared to the blank control group, DSS exposure significantly reduced Hcoepic cell viability by 26.7% (p<0.01). Compared to the DSS group, LOP-a, LOP-b, and LOP-t increased cell viability by 13.1% (p<0.05), 14.2% (p<0.05), and 21.6% (p<0.01), respectively. Under the same concentration conditions, LOP-t had a stronger inhibitory effect on DSS-induced reduction in intestinal epithelial cell viability than LOP-a, while LOP-b was used alone. We hypothesize that LOP-a and LOP-b in the combination may have a synergistic effect, thereby alleviating DSS damage to colonic epithelial cells.

[0057] The experimental results are shown in Figure 6 As shown in Figure A, compared to the blank control group (where intracellular ROS was 100%), the ROS content in Hcoepic cells after DSS stimulation was significantly higher than that in the blank control group by 231% (p<0.01), indicating that DSS exposure increased intracellular ROS content. Excessive ROS can induce cellular oxidative stress, damage cells, and reduce cell viability. After treatment with LOP-a, LOP-b, and LOP-t, the ROS content in Hcoepic cells decreased, and ROS generation was inhibited, indicating that LOP-a, LOP-b, and LOP-t inhibited DSS-induced ROS generation in Hcoepic cells. Compared to the DSS group, the intracellular ROS in the LOP-a, LOP-b, and LOP-t groups decreased by 41.4% (p<0.01), 38.4% (p<0.01), and 53.2% (p<0.01), respectively. Among them, the LOP-t group showed superior inhibition of DSS-induced ROS generation in Hcoepic cells compared to LOP-a and LOP-b at the same concentration. We hypothesize that the combined use of LOP-a and LOP-b has a synergistic effect, which can better inhibit the generation of ROS in Hcoepic cells caused by DSS exposure, thereby alleviating ROS-induced oxidative stress and oxidative damage to colonic epithelial cells.

[0058] Further analysis of the experimental results ( Figure 6B, 6 C) found that LOP-a, LOP-b, and LOP-t had a certain effect on the decrease in SOD activity and increase in MDA content of Hcoepic cells caused by DSS exposure. LOP-a, LOP-b, and LOP-t could increase the SOD activity of Hcoepic cells and inhibit the excessive secretion of MDA by Hcoepic cells caused by DSS exposure. Moreover, LOP-t had a greater effect on the SOD activity and MDA content of intestinal epithelial cells than LOP-a and LOP-b alone.

[0059] The experimental results are shown in Figure 7 As shown in A-F, compared to the blank control group (where the content of relevant inflammatory factors in the cell culture medium was 100%), the levels of inflammatory cytokines (IL-4, IL-5, IL-13, IFN-γ, TNF-α, IL-6) secreted by Hcoepic cells after DSS stimulation were significantly higher than those in the blank control group. This indicates that DSS exposure induces cellular inflammatory responses, thereby damaging cells and reducing cell viability. After treatment with horse LOP-a, LOP-b, and LOP-t, the levels of inflammatory cytokines (IL-4, IL-5, IL-13, IFN-γ, TNF-α, IL-6) secreted by Hcoepic cells decreased, and the production of inflammatory cytokines induced by DSS exposure was inhibited. Among them, the LOP-t group showed superior inhibition of DSS-induced inflammatory cytokine production activity compared to LOP-a and LOP-b at the same concentration. We hypothesize that the combined use of LOP-a and LOP-b has a synergistic effect, which can better inhibit the production of inflammatory cytokines induced by DSS exposure, thereby alleviating the inflammatory damage to colonic epithelial cells induced by DSS exposure.

[0060] Further analysis of the experimental results ( Figure 8(A-E) The study found that DSS exposure induced phosphorylation of NF-κB signaling pathway-related proteins in Hcoepic cells, activating the NF-κB signaling pathway. Further DSS exposure significantly increased the expression of NLRP3 inflammasome-related proteins (ASC, NLRP3, and Caspase-1) in Hcoepic cells, indicating that DSS exposure activated the NLRP3 inflammasome in Hcoepic cells. However, treatment with LOP-a, LOP-b, and LOP-t significantly inhibited the DSS-induced phosphorylation of NF-κB signaling pathway-related proteins and the increased expression of NLRP3 inflammasome-related proteins (ASC, NLRP3, and Caspase-1), indicating that DSS exposure-induced activation of the NLRP3 inflammasome and NF-κB signaling pathway in Hcoepic cells was suppressed. Among these, the LOP-t group showed superior inhibition of DSS-induced activation of the NLRP3 inflammasome and NF-κB signaling pathway in Hcoepic cells compared to LOP-a and LOP-b at the same concentration. We hypothesize that the combined use of LOP-a and LOP-b has a synergistic effect, which can better inhibit the activation of the NLRP3 inflammasome and NF-κB signaling pathway in Hcoepic cells induced by DSS exposure, thereby alleviating DSS exposure-induced colonic epithelial cell inflammation.

[0061] Further analysis of the experimental results ( Figure 9 (A-C) found that DSS exposure significantly reduced the expression of tight junction proteins in Hcoepic cells, while treatment with LOP-a, LOP-b, and LOP-t inhibited this reduction. Among these, the LOP-t group showed superior activity in inhibiting the reduction of Hcoepic cell tight junction protein expression induced by DSS exposure compared to LOP-a and LOP-b at the same concentration. We hypothesize that the combined use of LOP-a and LOP-b has a synergistic effect, better inhibiting the reduction of Hcoepic cell tight junction protein expression induced by DSS exposure, thereby alleviating DSS-induced inflammatory damage to colonic epithelial cells.

[0062] The above-disclosed embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of the invention. Those skilled in the art will understand that implementing all or part of the above-described embodiments and making equivalent changes in accordance with the claims of the present invention are still within the scope of the invention.

Claims

1. An active peptide, characterized in that, The amino acid sequence of the active peptide is shown in SEQ ID No. 1 or SEQ ID No.

2.

2. An active peptide composition, characterized in that, The active peptide composition comprises active peptides with the amino acid sequences shown in SEQ ID No. 1 and SEQ ID No.

2.

3. The active peptide composition according to claim 2, characterized in that, The mass ratio of the active peptides with the amino acid sequences shown in SEQ ID No. 1 and SEQ ID No. 2 is 1:1-10.

4. The active peptide composition according to claim 3, characterized in that, The mass ratio of the active peptides with the amino acid sequences shown in SEQ ID No. 1 and SEQ ID No. 2 is 1:1-5.

5. The active peptide composition according to claim 3, characterized in that, The mass ratio of the active peptides with the amino acid sequences shown in SEQ ID No. 1 and SEQ ID No. 2 is 1:

1.

6. The use of the active peptide or active peptide composition according to any one of claims 1 to 5 in the preparation of a medicament having an anti-inflammatory effect on colonic epithelial cells.

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