TWI863205B - Perch peptides having wound healing efficacy and composition thereof - Google Patents

Abstract

The disclosure relates to perch peptides having wound healing efficacy, and accordingly provides composition containing them, and the use for manufacturing a pharmaceutical composition or a food supplement for enhancing wound healing.

Description

Wound healing effects of perch peptides and their compositions

The present invention relates to a peptide having wound healing effect and a composition thereof.

The skin's primary function is to act as a protective barrier against the external environment and against excessive water loss. The skin is composed of two tissue layers: (1) the keratinized epidermis and (2) an underlying thick layer of collagen-rich connective tissue, the dermis, which provides support and nutrition. When the skin is injured, the body initiates a healing process to repair the injury. The wound healing process is a dynamic one that includes three highly integrated and overlapping phases: (1) an inflammatory response phase, which involves the extravasation of blood components leading to platelet aggregation, blood coagulation, and the migration of inflammatory cells to the wound site; (2) a proliferative phase, which involves the migration and proliferation of keratinocytes, fibroblasts, and endothelial cells, leading to re-epithelialization and granulation tissue formation; and (3) a tissue remodeling phase that restores tissue structural integrity and functional capacity. The entire process is coordinated and controlled by many cytokines and growth factors, which promote inflammatory cells and repair cells to heal wounds (Tang et al., A Small Peptide with Potential Ability to Promote Wound Healing, PLoS One. 2014; 9(3): e92082; March 19, 2014.).

Shigemura et al. found that after ingestion of collagen peptides, peptide substances will appear in peripheral blood vessels. Pro-Hyp, Pro-Hyp-Gly, Ala-Hyp, Ala-Hyp-Gly, Ser-Hyp, Ser-Hyp-Gly, Leu-Hyp, Ile-Hyp and Phe-Hyp have been identified as food-derived collagen peptides. Studies have suggested that food-derived collagen peptides may promote wound healing by stimulating fibroblasts and other cells to migrate to injured tissues (Shigemura et al., Effect of Prolyl-hydroxyproline (Pro-Hyp), a Food-Derived Collagen Peptide in Human Blood, on Growth of Fibroblasts from Mouse Skin. J Agric Food Chem. 2009; 57(2):444-9, 2018, January 28, 2009). A study by Yang et al. showed that low molecular weight peptides extracted from Alaska Pollock can be used as protein supplements and are potential active ingredients for promoting wound healing (Yang et al., 2018, Effects of oral administration of peptides with low molecular weight from Alaska Pollock ( Theragra chalcogramma ) on cutaneous wound healing. Journal of Functional Foods 2018, Vol. 48, Pages 682-691, September 2018). Therefore, low molecular weight peptides with wound healing effects have the potential to be used as drugs or food supplements to promote wound healing.

To date, there is still a need to develop peptides with wound healing effects for use in preparing pharmaceutical compositions or food supplements that promote wound healing.

Therefore, the present invention provides peptides having the effect of promoting wound healing, and compositions thereof, which can be used to prepare pharmaceutical compositions or food supplements for promoting wound healing.

On the one hand, the present invention provides a peptide having the effect of promoting wound healing, which has only one amino acid sequence selected from the group consisting of: FPSLVRGP (SEQ ID NO:1); GGLLCTHYNRLAV (SEQ ID NO:2); GALGMLGDYSLV (SEQ ID NO:3); LNLAMNALDLYL (SEQ ID NO:4); MGLLCKGSPATP (SEQ ID NO:5); TSEGTRVAPW (SEQ ID NO:6); QLGMLMYGPGLTGQ (SEQ ID NO:7); PEDVLLDAFKVLDPKYHRT (SEQ ID NO:8); MGLGTPWLNQF (SEQ ID NO:9); and GMTGLWPW (SEQ ID NO:10).

In a specific embodiment, the peptide is derived from sea bream and produced by enzyme hydrolysis. The peptide has the effect of promoting wound healing, and can be used to prepare a pharmaceutical composition or food supplement for promoting wound healing.

According to an embodiment of the present invention, the peptide has the ability to promote fibroblast crawling.

According to an embodiment of the present invention, the peptide has the effect of promoting the proliferation of fibroblasts.

According to an embodiment of the present invention, the peptide has the effect of promoting the secretion of fibronectin by fibroblasts.

According to an embodiment of the present invention, the peptide has the effect of promoting the secretion of collagen in fibroblasts.

In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a peptide or a combination thereof of the present invention and a pharmaceutically acceptable carrier.

In another aspect, the present invention provides a food supplement comprising an effective amount of a peptide or a combination thereof of the present invention and a carrier acceptable to food.

In a specific embodiment, the pharmaceutical composition or food supplement has the effect of promoting wound healing.

In yet another aspect, the present invention provides a use of a peptide for preparing a medicament for promoting wound healing.

In yet another aspect, the present invention provides a use of a peptide for preparing a food supplement for promoting wound healing.

It should be understood that the foregoing summary and the following detailed description are merely illustrative and explanatory and are not intended to limit the scope of the present invention.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one familiar with the art to which this invention belongs.

The present invention is further described by the following embodiments, which are intended to be illustrative rather than limiting.

As used herein, the article "a" or "an" refers to one or to more than one (ie, to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

As used herein, the term "subject" or "subject" includes humans and non-human animals, including companion animals (e.g., dogs, cats, etc.), farm animals (e.g., cows, sheep, pigs, horses, etc.), or experimental animals (e.g., rats, mice, guinea pigs, etc.).

According to the present invention, the preparation of the peptide of the present invention is to obtain the peptide stock solution from the sea bass extract, and further identify the following peptides with wound healing effect after hydrolysis, which have the following amino acid sequences: FPSLVRGP (SEQ ID NO:1); GGLLCTHYNRLAV (SEQ ID NO:2); GALGMLGDYSLV (SEQ ID NO:3); LNLAMNALDLYL (SEQ ID NO:4); MGLLCKGSPATP (SEQ ID NO:5); TSEGTRVAPW (SEQ ID NO:6); QLGMLMYGPGLTGQ (SEQ ID NO:7); PEDVLLDAFKVLDPKYHRT (SEQ ID NO:8); MGLGTPWLNQF (SEQ ID NO:9); and GMTGLWPW (SEQ ID NO:10).

According to the embodiments of the present invention, whether in animal models or cell models, it is verified that the peptide of the present invention has the effect of promoting wound healing, can quickly repair wound healing, and can promote the proliferation of fibroblasts.

According to the present invention, the peptides of the present invention can be obtained from organisms or artificially synthesized according to methods commonly known or used by those skilled in the art to which the present invention belongs, including, for example, separation and hydrolysis from sea bream extracts, or artificial synthesis according to amino acid sequences, or produced by recombinant gene technology.

The term "pharmaceutical composition" used herein refers to a composition comprising a therapeutically effective amount of the peptide of the present invention in combination with at least one pharmaceutically acceptable carrier. A person having ordinary knowledge in the field of the present invention can prepare the composition according to the relevant pharmaceutical standards and commonly used methods and procedures.

The peptide of the present invention can be prepared into different dosage forms as needed. For example, the peptide of the present invention can be prepared into oral dosage forms such as tablets, capsules, pills, powders, suspensions, syrups and emulsions as needed for administration. Or it can be prepared into an external dosage form and applied topically to a wound. It can also be prepared into an injection form for administration intravenously (concentrated injection or infusion), intraperitoneally, subcutaneously or intramuscularly. All of these forms use dosage forms familiar to those skilled in the medical field. It can be administered alone, but is usually administered together with a pharmaceutical carrier selected based on the selected route of administration and standard pharmaceutical practice.

The term "pharmaceutically acceptable carrier" used herein means a medium generally accepted by the pharmaceutical industry for delivering peptides into the body of a subject, including any known or customary pharmaceutically acceptable carrier, including adjuvants, excipients or vehicles, such as diluents, preservatives, fillers, flow regulators, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, aromas, lubricants and dispersants, depending on the nature of the mode of administration and the dosage form. Pharmaceutically acceptable carriers are formulated based on many factors that are within the knowledge or common scope of the art to which the present invention belongs, depending on the type and nature of the peptide to be formulated, the individual to be administered, the intended route of administration, and the therapeutic indication. Pharmaceutically acceptable carriers include aqueous and non-aqueous liquid media and various solid and semi-solid dosage forms. In addition to the active agent, such carriers may also contain various ingredients and additives, such as stabilizers, binders, etc.

The term "effective amount" as used herein means the desired regulation of the desired function using the peptide of the present invention. As noted below, the exact amount required will vary between individuals, depending on the individual's condition, age, sex, species and weight, the characteristics and formulation of the peptide, etc. The dosing regimen can be adjusted to induce the best therapeutic response. A person of ordinary skill in the art can determine the appropriate effective amount using routine experiments.

According to the present invention, a food supplement can also be prepared and used for individuals with wounds to promote wound healing by oral administration.

Food supplements can be prepared according to the general health food production process, with appropriate food additives and appropriate food-acceptable carriers added as needed.

The following non-limiting embodiments are helpful for those with ordinary knowledge in the art to which the present invention belongs to implement. Such embodiments should not be considered as limiting the present invention. Those with ordinary knowledge in the art to which the present invention belongs may modify and change the embodiments discussed herein without departing from the spirit or scope of the present invention, and still fall within the scope of the present invention.

Embodiment

Example 1 Preparation of the Peptide of the Invention

1.1 Preparation of sea bream essence

The perch extract is prepared according to the following steps: (1) removing the viscera of the perch and washing it with clean water to obtain a treated fish material; (2) placing the treated fish material in an appropriate amount of water and heating it under pressure to a temperature above 100°C for extraction; and then filtering and separating the perch extract.

1.2 Preparation of sea bream peptide stock solution

The following steps were followed to prepare the perch peptide stock solution: (1) the perch extract obtained in the above step was mixed with water at a ratio of 1:3 (w/v); (2) a composite enzyme was added to hydrolyze the mixture at 40°C to 60°C. The composite enzyme was purchased from Hengzhou Industrial Co., Ltd. and included composite protease, neutral protease, alkaline protease, papain, trypsin and exoprotease; (3) the mixture was cooled and filtered to obtain the perch peptide stock solution.

1.3 Sequence identification

1.3.1 LC-MS/MS analysis

The stock solution of sea bream peptide was diluted in HPLC buffer A (0.1% formic acid) and injected into a reverse phase chromatography column (Zorbax 300 SB-C18, 0.3×5 mm; Agilent Technologies). Then, a mobile phase gradient was performed on a self-made chromatographic column (Waters BEH 1.7 μm, 100 μm ID × 10 cm, 15 μm tip) using HPLC buffer B (99.9% acetonitrile/0.1% formic acid) at a flow rate of 0.3 μL/min within 70 minutes. The analysis conditions are shown in Table 1. Table 1: Chromatographic column analysis conditions time Buffer A (%)* Buffer B (%)** 0 95 5 5 88 12 42 72 27 52 64 36 57 52 48 58 43 57 62 43 57 64 95 5 70 95 5 *Buffer A is 0.1% formic acid in water. **Buffer B is 0.1% formic acid in acetonitrile.

The LC device was connected in series with a 2D linear ion trap mass spectrometer (Orbitrap Elite ETD; Thermo Fisher) operated with Xcalibur2.2 software (Thermo Fisher). The electrospray voltage was set to 2.0 kV and the capillary temperature was set to 200 °C.

1.3.2 Peptide Identification (De novo Sequencing)

Data analysis was performed using PEAKS studio (version 10.5, Bioinformatics Solutions Inc.). MS/MS spectra were analyzed using the de novo sequencing function.

The peptide identification conditions were as follows: 10 ppm mass tolerance was allowed for intact peptide mass, 0.1 Da was allowed for HCD fragment ions; oxidation of methionine, acetyl (protein N-terminus) and deamination (asparagine) were used as variable modifications. Peptide matching spectra (PSM) were filtered based on an average local confidence (De novo score) of 50%.

2. Results

2.1 Peptide sequence identification

The peptide sequences in the effective perch peptide stock solution were analyzed to obtain novel amino acid fragments, as shown in Table 2. Table 2: Sequences and relative proportions of the peptides of the present invention Peptide sequence Relative content FPSLVRGP (SEQ ID NO: 1) 6.21% GGLLCTHYNRLAV (SEQ ID NO:2) 5.16% GALGMLGDYSLV (SEQ ID NO:3) 2.89% LNLAMNALDLYL (SEQ ID NO:4) 2.87% MGLLCKGSPATP (SEQ ID NO:5) 2.79% TSEGTRVAPW (SEQ ID NO:6) 2.66% QLGMLMYGPGLTGQ (SEQ ID NO:7) 1.99% PEDVLLDAFKVLDPKYHRT (SEQ ID NO:8) 1.90% MGLGTPWLNQF (SEQ ID NO:9) 1.72% GMTGLWPW (SEQ ID NO: 10) 1.49%

Example 2 Efficacy Experiment

2.1 Animal Mode

2.1.1 Source and breeding of experimental animals

Sprague-Dawley (SD) male rats aged 8 weeks were purchased from the Lesco Animal Center. The animals were housed in the rat area of the GLP animal laboratory of our hospital. The lighting was adjusted to 12 hours day/12 hours night, the positive pressure environment, the temperature was maintained at 22±4℃, and the humidity was maintained at 30%-70%. The experiment adopted the principle of free food and water. The animals were fasted for 16±2 hours the day before the experiment, and feed was provided after the experiment was completed.

2.1.2 Model establishment

Open wounds:

Rats were anesthetized with Zoletil50 (50 mg/kg) injected intraperitoneally, and Ketoprofen (3 mg/kg) was used for analgesia. The hair around the wound site was shaved with scissors, wiped with iodine and then deiodinated with 75% alcohol. A circular mark with a diameter of about 2 cm was drawn on the back of the rat, and then a sterile instrument was used to make a wound to the depth of the dermis.

Pain Relief:

Ketoprofen (3 mg/kg) was injected intramuscularly for 3 consecutive days after incision to relieve pain.

Dressing changes:

Three hours after oral administration of the test substance, the wound was cleaned with physiological saline, dried with sterile gauze, and then covered with 3M Tegaderm waterproof breathable patch and sterile gauze, and finally bandaged with elastic bandage. The dressing was changed and re-bandaged on days 0, 1, 2, 3, 5, 7, 9, 12, 15, 18, and 21 after wound incision.

2.1.3 Dosage and grouping of perch peptides

The perch peptide combination of the present invention was used for the experiment, and the groups were as shown in Table 3. Table 3: Grouping of Animals in Wound Healing Experiment Group Dosage Injection volume Number of animals Control - Each rat was orally administered 1.8 mL of water for injection 3 Sea bass peptide low dose group (BL) 0.585 g/kg When using, add 6 mL of injection water to each tube to reconstitute, and each rat is orally administered 1.8 mL 3 Sea bass peptide medium dose group (BM) 1.17 g/kg 3 Sea bass peptide high dose group (BH) 2.925 g/kg 3

2.1.4 Animal experiments on wound healing

The wound healing animal experiment was divided into groups according to Table 4, including the peptide of the present invention and the control group. Table 4: Wound healing animal experiment grouping Group Dosage Injection volume Number of animals Control - Each rat was orally administered 2.0 mL of water for injection 8 The peptide group of the present invention 1.972g/kg Add 16.0 mL of injection water to each tube for reconstitution, and orally administer 2.0 mL to each rat. 8

2.1.5 Observation and analysis items

The following observations, measurements and analyses were performed on the rats:

Body weight measurement: The body weight of rats was recorded before incision and once a week after incision.

Clinical symptom observation: Observe at least once a day, including mental state, behavior, respiratory status, mouth, nose, eyes, appearance and skin.

Wound healing observation and calculation: On days 0, 1, 2, 3, 5, 7, 9, 12, 15, 18, and 21, take photos and save them (with a scale sticker attached to the wound for calibration), trace them with transparent slides, and then scan them into images. Use Image J software to analyze and calculate the wound healing area, and then calculate the wound healing efficiency percentage (%).

2.2 Cell Model

2.2.1 Fibroblast cell crawling test

This test uses human fibroblasts as the test platform. The cells are inoculated into the Oris ^TM migration kit. After removing stoppers according to the supplier's instructions, the test substances are added for 18 ± 2 hours. Then, fluorescent dye (Calcein AM) is added to mark the cells. The degree of cell crawling into the detection zone is quantitatively compared by detecting the fluorescence intensity (ex/em: 485/528 nm). Formula = [value of sample treatment group - blank control group] / [negative control group - blank control group] x 100% Blank control group: the initial state of cells without crawling; Negative control group: the basic value of cell movement in the group without sample treatment is used as the benchmark for quantitative comparison 100%; Positive control group: (5 μg/mL EGF) crawling ability must be ≧ 120%.

2.2.2 Fibroblast proliferation test

This test uses human fibroblasts as the test platform. The cells are cultured with different test substances (unfiltered) in serum-free medium for 24 and 48 hours (5±1% CO ₂ , 37±1°C). The cell morphology is observed under an inverted microscope (qualitative analysis), and the activity is analyzed by MTS/PMS reagents and cell action assays (quantitative analysis) to evaluate whether the test samples have the ability to promote fibroblast proliferation.

2.2.3 Promoting interstitial secretion test

Human skin fibroblasts were seeded at a density of 2x10 ⁵ in a 6-well plate. After cell attachment, the culture medium was replaced with a base medium (DMEM medium) without fetal bovine serum and the test substances were added. The cells were cultured in a humidified 5% carbon dioxide incubator at 37°C for 2 days. The supernatant was collected and quantitatively detected by enzyme immunoassay for fibronectin (R&D) and pro-collagen I (R&D). The above detection operation was performed according to the instructions of each detection kit. The absorbance value at a wavelength of 450 nm was measured by a microplate analyzer, and then the content in the supernatant was calculated by applying the concentration relative to the absorbance value calibration curve. The negative control group (NC) was given DMEM medium, and the positive control group (PC) was given 5% fetal bovine serum. The experiment was repeated three times.

2.3 Statistical analysis

The experimental data were repeated three times, and the results were expressed as mean ± SD. The wound healing rate was analyzed by One-Way ANOVA. If p < 0.05, it means that there is a significant difference between at least two groups. Duncan post hoc test was then used to analyze whether there is a significant difference between the data of each group (p < 0.05).

2.4 Results

2.4.1 Animal Wound Healing Experiments

Experimental results: The experimental results are shown in Figure 1. Day 7: The high-dose group of sea bream peptide (B-H) healed faster, with a significant difference compared to the control group (p < 0.05); Day 9: The high-dose group of sea bream peptide (B-H) healed faster, with a significant difference between the high-dose group of sea bream peptide (B-H) and the control group (p < 0.05). Overall healing effect ranking: high-dose group of sea bream peptide (B-H) > medium-dose group of sea bream peptide (B-M) > low-dose group of sea bream peptide (B-L) > control group. From the above results, it can be seen that the sea bream peptide of the present invention has a good effect on wound healing. The results on the 12th day showed that the healing effect was better with medium dose or above through gavage, which was a dose-effect.

2.4.2 Cell crawling experiment - sea bream peptide

The experimental results are shown in Figure 2. At a 400-fold dilution concentration, the crawling ability of fibroblasts in the perch peptide stock solution was 121.1%, which was better than that of the negative control group, indicating that the perch peptide of the present invention has the ability to promote the crawling of skin fibroblasts and also has a certain effect on wound healing.

2.4.3 Fibroblast proliferation assay

The experimental results are shown in FIG3 . The activity growth rate of the sea bream peptide group of the present invention at various dilution concentrations is higher than that of the negative control group, indicating that the sea bream peptide of the present invention has a positive effect on the proliferation of fibroblasts.

2.4.4 Promoting interstitial secretion test

The experimental results are shown in Figure 4. All samples have a certain effect in promoting the secretion of fibronectin. Among them, the perch peptide has a positive correlation with the secretion of fibronectin as the concentration increases.

2.4.5 Promoting interstitial secretion test

The experimental results are shown in Figure 5. The test for promoting pro-collagen I secretion showed that when fibroblasts were treated with a specified concentration of the sample, the increase in the concentration of perch peptide was positively correlated with the secretion of pro-collagen I.

2.4.6 Animal Wound Healing Experiment - Perch Peptide

The experimental results are shown in Figure 6. The wound healing efficiency of the perch peptide group showed significant differences compared with the control group starting from the second day, and the significant differences continued to be shown on the third, fifth, and seventh days.

In summary, it is shown that the sea bass peptide or its combination of the present invention has the effect of promoting wound healing and can be developed into wound healing medicine or food supplement.

without

When read in conjunction with the accompanying drawings, the foregoing invention content and the following detailed description of the invention will be better understood. In order to illustrate the present invention, the currently preferred specific embodiments are shown in the accompanying drawings.

Figure 1 shows the results of wound healing efficiency (%) of rats after taking different doses of the peptide of the present invention.

FIG2 shows the quantitative analysis of different doses of the peptide of the present invention and the negative control group and the positive control group in the cell crawling experiment.

FIG3 shows the effects of different doses of the peptide of the present invention and the negative control group on promoting the proliferation of fibroblasts after being co-cultured with fibroblasts for 24 and 48 hours.

FIG4 shows the comparison of different doses of the peptide of the present invention and the negative control group in promoting the secretion of fibronectin by fibroblasts after 48 hours of co-culture with fibroblasts. The data marked with "*" in the figure represent significant differences between them and the control group, "*" means P <0.05;"****" means P < 0.0001.

FIG5 shows the comparison of different doses of the peptide of the present invention and the negative control group in promoting the secretion of collagen in fibroblasts after 48 hours of co-culture with fibroblasts. The data marked with "*" in the figure represent significant differences between them and the control group, "*" indicates P <0.05;"****" indicates P < 0.0001.

FIG6 shows a comparison of the wound healing efficiency (%) of the peptide of the present invention and the control group on rats. The data marked with "*" in the figure represent significant differences between the data and the control group, and "*" indicates P < 0.05.

without

TWI863205B_112112677_SEQL.xml

Claims (10)

A composition having wound healing efficacy, comprising a combination of peptides having the following amino acid sequences: FPSLVRGP (SEQ ID NO: 1); GGLLCTHYNRLAV (SEQ ID NO: 2); GALGMLGDYSLV (SEQ ID NO: 3); LNLAMNALDLYL (SEQ ID NO: 4); MGLLCKGSPATP (SEQ ID NO: 5); TSEGTRVAPW (SEQ ID NO: 6); QLGMLMYGPGLTGQ (SEQ ID NO: 7); PEDVLLDAFKVLDPKYHRT (SEQ ID NO: 8); MGLGTPWLNQF (SEQ ID NO: 9); and GMTGLWPW (SEQ ID NO: 10). The composition having wound healing effect as described in claim 1, wherein the peptides are derived from perch. The composition having wound healing effect as described in claim 1, wherein the peptide is hydrolyzed by enzyme. The wound healing composition as described in any one of claims 1-3 has the ability to promote fibroblast crawling. The wound healing composition as described in any one of claims 1-3 has the effect of promoting fibroblast proliferation. The wound healing composition as described in any one of claims 1-3 has the effect of promoting the secretion of fibronectin by fibroblasts. The wound healing composition as described in any one of claims 1 to 3 has the effect of promoting collagen secretion of fibroblasts. A pharmaceutical composition comprising a therapeutically effective amount of a composition having wound healing efficacy as described in any one of claims 1 to 3, and a pharmaceutically acceptable carrier. A food supplement comprising an effective amount of a composition having wound healing effect as described in any one of claims 1 to 3, and a carrier acceptable to food. A composition having wound healing efficacy as described in any one of claim items 1-3 is used for preparing a medicine for promoting wound healing.