Patent Application
20240279276
The present invention relates to the technical field of bioactive peptides, and in particular to an anti-aging salmon roe tripeptide and a preparation method and application thereof.
Skin aging is the external manifestation of human aging. A mechanism of aging is complex. Studies in recent years have shown that the pathogenesis may be related to many factors, including lipid metabolism, oxidative stress, inflammation, apoptosis, etc. Studies have shown that the main reason for skin aging is that the oxygen free radicals produced by ultraviolet rays and other factors affect the normal growth cycle of skin cells, promote the hydrolysis of collagen and other extracellular matrix by protease, and cause DNA damage and the reduction of matrix protein synthesis, thereby resulting in skin relaxation and reduction of elasticity. With the stimulation of facial muscle fibers by expressions, the most significant sign of skin aging, i.e., wrinkles are produced.
Mechanism of endogenous skin aging: endogenous aging of skin is an irreversible and slow physiological process. Endogenous skin aging is evident only after a certain stage of age, and is characterized by skin drying, roughness, elasticity reduction, and the production of wrinkles. With the increase of age, the dermal mast cells and fibroblasts in the skin tissue are decreased, the secretion of collagen is decreased, and the dermo-epidermal junction area is flattened. Under the influence of aging of other organs in a human body, endogenous skin aging is caused by many factors. From the perspective of the physiological mechanism, oxidative stress causes damage to DNA, protein and other components of cells, and exacerbates progressive telomere shortening, which is an important reason for endogenous skin aging.
Exogenous skin aging: exogenous factors, such as ultraviolet irradiation, pollution and smoking, produce a series of reactions such as pro-oxidation/anti-oxidation through neuroendocrine immune regulation, and affect cell renewal, so as to permanently affect the physiological function of the skin. Ultraviolet induced photoaging is the currently recognized most important cause of skin aging.
Reactive oxygen species (ROS) produced by UV induction can damage DNA and inhibit tyrosine phosphatases, which leads to signal transduction enhancement and ultimately leads to up-regulation of the transcription factor AP-1. At the same time, ultraviolet ray can also lead to the up-regulation of c-Jun as one of the components of AP-1, and down-regulation of retinoic acid receptors, to further weaken the inhibitory effect of retinoic acid on AP-1. Moreover, ultraviolet ray directly induces DNA variation, up-regulates nuclear factor-kB (NF-kB), and inhibits transforming growth factor-β (TGF-β)-mediated cell signaling pathways. These effects lead to degradation or secretion decrease of collagen. Collagen as the most widespread matrix protein of the body provides the support and elasticity for skin. Once the balance of collagen secretion/degradation is broken, the content of collagen in the skin will be reduced, thereby affecting the stability of the skin structure. With the excessive stimulation of the fiber by muscle movement such as expression muscle, the most significant sign of skin aging, i.e., wrinkles are produced. Therefore, the synergistic use of active ingredients with multiple action mechanisms is a preferred choice to achieve a desired anti-aging effect.
Functional extracts separated from aquatic food can be used as functional food and nutritional health products. Peptides obtained from aquatic protein by biotechnology means not only show high nutritional value, but also show biological characteristics for diet or therapeutic purposes, have special active aquatic extracts, and may become functional food for human nutrition. Fish is the earliest biological resource that people begin to eat and rich in protein, vitamins and minerals, and is a high-quality raw material for the development of functional foods such as oligopeptide. At present, a large number of antioxidant, antitumor, antibacterial and anti-inflammatory polypeptides have been obtained by enzymolysis and separation from silver carp, Pacific saury, tilapia and the like, and new structures and mechanisms of action have been continuously discovered. Therefore, rational and efficient development and utilization of freshwater fish resources has broad market prospects, and has certain social and economic significance.
As a kind of popular edible fish, salmon is rich in nutrients, especially Ω3 fatty acids, and is beneficial to heart health and a brain function. Therefore, deep processing of the salmon can not only make full use of resources and achieve high-value utilization, but also increase an added value of the industry and promote sustainable and healthy development of the salmon aquaculture industry.
However, there is no report on use of the salmon roe polypeptide for the anti-aging efficacy.
Therefore, how to provide a salmon roe polypeptide with an anti-aging efficacy is a problem needing to be solved urgently by those of skill in the art.
In view of this, a purpose of the present invention is to provide an anti-aging salmon roe tripeptide and a preparation method and application thereof, so as to solve the defects in the prior art.
In order to achieve the above purpose, the present invention employs the following technical solution:
An anti-aging salmon roe tripeptide is provided, having a peptide sequence of YLP.
A preparation method of the anti-aging salmon roe tripeptide specifically includes the following steps:
Further, in step (1), a mass ratio of the salmon roe to the deionized water is 1:6.
Further, in step (1), for homogenization, a rotating speed is 8000 rpm, and a time is 1 min.
Further, in step (2), an addition amount of the pancreatin is 1%.
Further, in step (2), for enzymolysis, a temperature is 55° C., and a time is 4 h.
Further, in step (2), for enzyme inactivation, a temperature is 90-100° C., and a time is 10-15 min.
Further, in step (2), cooling is performed until a room temperature is reached.
Further, in step (3), for centrifugation treatment, a temperature is 4° C.; a rotating speed is 8000 rpm; and a time is 15 min.
Further, in step (3), a storage temperature is −80° C.
The present invention further requests the protection of application of the salmon roe tripeptide or the salmon roe tripeptide prepared by the above preparation method in the preparation of an anti-aging drug, anti-aging foods, an anti-aging health care product or anti-aging cosmetics.
It can be seen from the above technical solution that compared with the prior art, the present invention has the following beneficial effects:
By evaluating a content of proteins, a degree of hydrolysis, a protein recovery rate, an ABTS antioxidant activity and a tyrosinase inhibitory activity of a salmon roe peptide, the present invention screens preparation conditions with a solid-liquid ratio being 1:6, the addition amount of an enzyme being 1% (according to a mass of a substrate), the enzymolysis time being 4 h, the temperature being 55° C., and the pancreatin selected as a salmon roe peptide-based delivery system; and a content of lipids of the salmon roe peptide is lower than that of an enzymolysis product of salmon, but a content of polyunsaturated fatty acids have an advantage.
A salmon roe peptide prepared by the present invention can increase levels of oxidative stress and skin-related factors (type I collagen and hyaluronic acid) by regulating a cell proliferation state, thereby achieving the protective effect on skin cells on the cell level.
It can be seen from results of an animal experiment, the salmon roe prepared by the present invention can achieve the anti-aging effect by regulating the oxidative stress and a skin state in an aging small water body.
Based on virtual screening means, the present invention finds that the anti-aging tripeptide YLP derived from the salmon can play an important anti-aging role in the body, and has high absorption efficiency, which can play an important role in the body, at the same time.
Technical solutions in the embodiments of the present invention are described clearly and fully below. Apparently, the described embodiments are merely part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those ordinary skilled in the art without contributing creative labor will belong to the protection scope of the present invention.
A preparation method of an anti-aging salmon roe tripeptide specifically includes the following steps:
A preparation method of an anti-aging salmon roe tripeptide specifically includes the following steps:
A preparation method of an anti-aging salmon roe tripeptide specifically includes the following steps:
This research aims to optimize hydrolysis conditions of the salmon roe, so as to obtain a bioactive peptide. An experimental design covers changes of different enzyme types, enzymolysis times, enzyme dosages and solid-liquid ratios.
First, 10 g of the salmon roe is homogenized with ultrapure water at 8000 rpm for 1 min. Then, a resultant is regulated to an optimum pH value, an enzyme is added, and a sample is placed in a constant temperature oscillator for hydrolysis at 55° C. Subsequently, the sample is placed in a boiling water bath for enzyme inactivation at 90-100° C. for 10-15 min. After the sample is cooled to the room temperature, the sample is centrifuged at 4° C. and 8000 rpm for 15 min; and a supernatant is taken to be used for measurement on subsequent indexes, and then stored at −80° C. for later use.
Investigation with single factor such as the enzyme type: in a single factor research in terms of the enzyme type, the addition amount of the enzyme is 1%; the hydrolysis time is 4 h; and the solid-liquid ratio is 1:6. Influences of different proteases (pancreatin, Alcalase, and a complex enzyme at a ratio of the pancreatin to the Alcalase being 1:1) on a hydrolysis effect are tested.
Investigation with single factor such as the enzymolysis time: another single factor research aims to determine the influence of the enzymolysis time on formation of a peptide. Under the condition with the solid-liquid ratio being 1:6 and the addition amount of the enzyme being 1%, the pancreatin is used for hydrolysis, and the hydrolysis times of 2 h, 4 h, 8 h, 12 h and 16 h are set separately.
Investigation with single factor such as the addition amount of the enzyme: the influence of the enzyme dosage on a hydrolysis effect is evaluated by means of a single factor research. The solid-liquid ratio is maintained at 1:6, the pancreatin is used for hydrolysis for 4 h, and the dosages of the enzyme added are 0.2%, 0.5%, 0.8%, 1%, and 2% separately.
Investigation with single factor such as the solid-liquid ratio: finally, the influence of the solid-liquid ratio on the hydrolysis effect is explored by means of a single factor research. Salmon roe samples are mixed with 20 mL, 40 mL and 60 mL of water, respectively, and 1% pancreatin is added for hydrolysis for 4 h.
The degree of hydrolysis is measured by an OPA method, and the contents of the proteins in the salmon roe and an enzymolysis supernatant are measured by a Kjeldahl method in GB 5009.5-2016 National Food Safety Standard-Determination of Protein in Foods.
Protein recovery rate %=content of proteins in enzymolysis supernatant/content of proteins in salmon roe×100%.
A measurement method for an ABTS free radical scavenging ability is an experimental method for evaluating an antioxidant capacity of a compound or a sample. ABTS (2, 2′-azino-bis(3-ethylbenzothiazoline) is a synthetic free radical compound that can be used for simulating oxygen free radicals in an organism. The method judges the antioxidant properties by measuring the ABTS free radical scavenging ability of the compound. During measurement, a color of ABTS free radicals will be changed, and addition of an antioxidant substance will lead to fading, and the degree is proportional to the antioxidant capacity. The method includes the basic steps:
In this experiment, the ABTS free radical scavenging ability of the enzymolysis supernatant is measured by referring to an optimized method.
L-tyrosine solution: 0.025 g of L-tyrosine is weighed, and added to 50 mL of sterile ionic water; a mixture is dissolved with a phosphate buffer solution with a volume fixed at 50 mL; and the L-tyrosine solution is prepared when needed.
Tyrosinase solution: the tyrosinase is prepared with the phosphate buffer solution until 1000 U/mL; and the tyrosinase solution is stored in the dark at −20° C., and is prepared when needed.
Sample solution: the sample is diluted to a concentration of 1 mg/mL with PBS buffered saline; and the sample solution is prepared when needed.
Referring to amounts of reagents added in Table 1, the L-tyrosine solution, the sample solution/reagent, and the phosphate buffer solution are sequentially added to a reaction system, and fully mixed; a mixture is incubated at a constant temperature of 37° C. for 10 min; then 20 L of the tyrosinase solution is added to wells sequentially for uniform mixing and a reaction at 37° C. for 5 min; a resultant is immediately put into a microplate reader; and the absorbance is tested at 475 nm.
The degrees of hydrolysis and the protein recovery rates according to the enzyme types are shown in
It can be seen from
The ABTS free radical scavenging activities according to the enzyme types are shown in
It can be seen
Tyrosinase inhibitory activities according to the enzyme types are shown in
It can be seen
The protein recovery rates and the degrees of hydrolysis under the enzyme hydrolysis times are shown in
It can be seen
The ABTS free radical scavenging activities under the enzyme hydrolysis times are shown in
It can be seen
Tyrosinase inhibitory activities under the enzyme times are shown in
It can be seen
The degrees of hydrolysis and the protein recovery rates at addition amounts of enzyme are shown in
It can be seen
The ABTS free radical scavenging activities at addition amounts of enzyme are shown in
It can be seen
Tyrosinase inhibitory activities at addition amounts of enzyme are shown in
It can be seen
The degrees of hydrolysis and the protein recovery rates at solid-liquid ratios are shown in
It can be seen
The ABTS free radical scavenging activities at the solid-liquid ratios are shown in
It can be seen
Tyrosinase inhibitory activities at the solid-liquid ratios are shown in
It can be seen
A 250 mL flat-bottomed flask has a constant weight and is weighed. A sample is weighed and added to a 50 mL colorimetric tube, and 2 mL of 95% ethanol and 4 mL of water are added for uniform mixing. 10 mL of a hydrochloric acid solution is added for uniform mixing. The colorimetric tube is put into a water bath at 70-80° C. for hydrolysis for 40 min. The flask is shaken every 10 min, to make particles adhered to a wall of the flask mixed into the solution. After hydrolysis is completed, the colorimetric tube is taken out, and cooled to the room temperature.
10 mL of the 95% ethanol is added for uniform mixing. 30 mL of an ether/petroleum ether mixed solution is added, and the colorimetric tube is covered. The colorimetric tube is shaken for 5 min, and left for standing for 10 min. An ether layer extract is collected into a 250 mL flask. A hydrolysate is repeatedly extracted for 3 times according to the above steps, the ether layer is evaporated to dryness in a water bath, residues are a fat extract. The flask is put into an oven at 60° C. for drying until a constant weight, and is weighed.
Contents of fats in the salmon roe and a freeze-dried enzymatic hydrolysate are shown in Table 2.
4 mL of 0.5 mol/L sodium methoxide is added to a flask, and heated in a water bath at 45° C. for 20 min; a sample solution in the flask is transferred into a 20 mL colorimetric tube; 4 mL of a 14% trifluoro(methanol)boron solution is added; and a mixture is heated in a water bath at 45° C. for 20 min. The mixture is cooled to the room temperature, 3 mL of n-hexane is added for extraction for 2 min; a resultant is left for standing for laying; and then an n-hexane layer is for later test.
An initial injection temperature is 100° C., maintained for 13 min, raised to 180° C. at 10° C./min, maintained for 6 min, raised to 192° C. at 1° C./min, maintained for 9 min, raised to 240° C. at 4° C./min, and maintained for 2 min. Operating conditions of a chromatographic column are as follows: an injection volume is 1 μL; a split ratio is 20:1; an injection temperature is 260° C.; a temperature of an ion source is 240° C.; and a SCAN full scan mode is used with a scanning range of 40-400. Results are expressed by g/100 g.
Results of unsaturated fatty acids in the salmon roe and the enzymatic hydrolysate are shown in Table 3.
By evaluating the content of the proteins, the degree of hydrolysis, the protein recovery rate, the ABTS antioxidant activity and the tyrosinase inhibitory activity of the salmon roe peptide, preparation conditions with the solid-liquid ratio being 1:6, the addition amount of the enzyme being 1% (according to a mass of a substrate), the enzymolysis time being 4 h, the temperature being 55° C., and the pancreatin selected as a salmon roe peptide-based delivery system are screened.
HaCaT cells in a logarithmic growth phase are taken; a concentration of the cells is regulated to 1.5×104 cells/mL; the cells are inoculated to a 96-well plate, and divided into a normal group and sample groups with different concentration gradients; the normal group is given a blank culture medium; six parallel wells are set for each concentration of a salmon roe extract (with the concentrations of the proteins are 0.1 mg/mL, 0.2 mg/mL, 0.5 mg/mL, 1 and 2 mg/mL respectively); culture is performed for 24 h; and an OD value of each group is measured by a CCK-8 method, and a relative cell viability is calculated.
The HaCaT cells are inoculated to the 96-well plate at a concentration of 1.5×104 cells per well, with 100 μL per well; adherent culture is performed in an incubator at 37° C. and with 5% CO2 for 24 h; and a culture medium is sucked and discarded. The cells are divided into a normal group and H2O2 damage groups (0.5 mmol/L, 0.6 mmol/L, 0.7 mmol/L, 0.8 mmol/L, 0.9 mmol/L, 1 mmol/L, 1.1 mmol/L, 1.2 mmol/L, and 1.3 mmol/L); six replicates are set for each group and incubated in 5% CO2 at 37° C. for 2 h; and an OD value of each group is measured by the CCK-8 method, and a relative cell viability is calculated.
2.2 Research on Protective Effects of Samples on HaCaT Cells with H2O2 Damage
The HaCaT cells are inoculated to a 96-well plate at a concentration of 1.5×104 cells per well, with 100 μL per well; adherent culture is performed in an incubator at 37° C. and with 5% CO2 for 24 h; and a culture medium is discarded. The cells are divided into a normal group, a model group, and an administration group. The normal group and the model group are given a blank culture medium; and different concentration gradients of samples are added to the administration group. Six replicates are set for each group, and incubated in 5% CO2 at 37° C. for 24 h; and a supernatant is sucked and discarded. Except that the blank culture medium is added to the normal group, 900 μmol/L H2O2 is added to both the model group and the administration group for a reaction for 6 h. An OD value of each group is measured by the CCK-8 method, and a relative cell viability is calculated.
The HaCaT cells are inoculated to a 6-well plate at a concentration of 6×105 cells per well; adherent culture is performed in an incubator at 37° C. and with 5% CO2 for 24 h; and a culture medium is discarded. The cells are divided into a normal group, a model group, and an administration group. The normal group and the model group are given a blank culture medium; and different concentration gradients of samples are added to the administration group. The cells are incubated in 5% CO2 at 37° C. for 24 h; and a supernatant is sucked and discarded. Except that the blank culture medium is added to the normal group, 900 μmol/L H2O2 is added to both the model group and the administration group for a reaction for 6 h. The cells are scraped down with a cell scraper, and broken for later use.
300 g of 1.0×106 cells are transferred to a 5 mL flow tube, centrifuged at the room temperature for 5 min; a centrifugate is removed; 2 mL of a binding buffer is added; 300 g of a mixture is centrifuged at the room temperature for 5 min; and a supernatant is removed. The cells are resuspended with 100 μL of a stain buffer; and 5 μL of Annexin V APC and 5 μL of a PI solution are added for incubation at the room temperature for 25 min. 100 μL of the stain buffer is added for computer detection.
A cell treatment method is consistent with that in “2.3 Apoptosis detection”. The cells are scraped down with a cell scraper, and broken for later use. Indexes are detected complying with operations of steps of relevant kits for GSH (glutathione), SOD (superoxide dismutase), MDA (malondialdehyde) and LDH (lactate dehydrogenase).
HaCaT (HDF human dermal fibroblasts inoculated to wells with 1×106 per well) human immortalized epidermal cells are inoculated with 1.2×106 per well. After cell adherence, 200 μL of a gun head is used to draw a straight line in a 6-well plate. The cells are divided into a normal group, a 0.4 mg/mL (the concentration of the proteins) salmon roe extract, and a 0.9 mg/mL (the concentration of the proteins) salmon roe extract. Abasic culture medium is added to the normal group, and sample solutions prepared by the basic culture medium are added to the sampling groups. Cell growth is observed at 0 h, 12 h and 24 h respectively.
HaCaT human immortalized epidermal cells and HDF human dermal fibroblasts are inoculated to a 12-well plate at 3×105 per well. After 24 h of cell adherence, normal culture medium/samples (with two concentration gradients set) are added. After 24 h of culture, a culture supernatant is collected for later test. (ANanjing Jiancheng human hyaluronic acid (HA) enzyme-linked immunosorbent assay kit, and a human type I collagen (Col I) enzyme-linked immunosorbent assay kit).
Modeling concentration screening is shown in
It can be seen from
2.7.2 Research on Protective Effects of Enzymolysis Products of Salmon Roe on HaCaT Cells with H2O2 Damage
Results are shown in
The cytotoxicity of the salmon enzymatic hydrolysate is shown by A in
It can be seen from A in
The protective effects of the enzymolysis product of the salmon roe on the HaCaT cells with H2O2 damage are shown by B in
It can be seen from B in
2.7.3 Protective Effects, Analyzed by Flow Cytometer, of Samples on HaCaT Cells with H2O2 Damage
Results are shown in Table 4 and
It can be seen from Table 4 that different quadrants of the flow cytometer represent different states of the cells.
It can be seen from
Results are shown in
As a coenzyme of a variety of enzymes such as GSH-Px, GSH is involved in removal of ·HO, O2−, H2O2, etc., and can effectively protect a body from being damaged by oxidative stress. Therefore, an amount of the GSH can reflect the body's antioxidant capacity to a certain extent.
The influences of the salmon enzymatic hydrolysates on a level of the glutathione (GSH) in the damaged cells are shown from Ain
It can be seen from A in
SOD can convert harmful superoxide free radicals to H2O2 by means of cellular respiration. A cell damage can lead to a change on the oxidative stress level. In order to further explore the reason, a project team detects an activity of the superoxide dismutase (SOD) in the cells.
The influences of the salmon enzymatic hydrolysates on the superoxide dismutase (SOD) in the damaged cells are shown by B in
It can be seen from B in
It can be seen from results that the salmon roe extract can achieve the purpose of improving the oxidative stress state of the cells by improving the activity of the SOD.
Malondialdehyde (MDA) is one of symbolic products of lipid peroxidation, and the content in the organism can directly reflect an oxidation or damage degree of the organism. Therefore, improvement in MDA level of the body is also one of important means to achieve anti-oxidation and maintain cell viability.
The influences of the salmon enzymatic hydrolysates on the malondialdehyde (MDA) in the damaged cells are shown by C in
It can be seen from C in
Destruction of cell membrane structures caused by apoptosis or necrosis will lead to release of enzymes in cytoplasm into a culture fluid, wherein enzymes include the lactate dehydrogenase (LDH) with a stable enzyme activity. Quantitative analysis on the cytotoxicity can be achieved by detecting the activity of the LDH.
The influences of the salmon enzymatic hydrolysates on the lactate dehydrogenase (LDH) in the damaged cells are shown by D in
It can be seen from D in
Based on the above experimental results, the concentration of the proteins in the salmon roe extract is selected as 0.9 mg/mL for a cell scratch test.
HDF cells are an abbreviation of human dermal fibroblast cells. It is a cell type found in a dermis layer of human skin. It is mainly responsible for synthesis of collagen, elastic fibers and other extracellular matrix molecules to maintain a structure, elasticity and stability of the skin. The HDF cells play an important role in physiological and pathological processes such as skin healing, wound repair, and skin aging. Due to the key role in skin health and diseases, researchers often use the HDF cells to explore cell biology, molecular mechanisms, and pathogenesis of related diseases.
The HaCaT cells are a human skin epithelial cell line, also known as human keratinocyte cells (HaCaT). This cell line is separated and cultured from normal human skin keratoma. The HaCaT cells have a high proliferative activity in in vitro culture, and can be divided continuously to form multi-layer cell accumulation, which is similar to an epidermal layer of the human skin. As they can simulate the characteristics of skin epithelial cells, the HaCaT cells are often used in a skin biological research, such as research on cell proliferation, differentiation, keratogenesis, or skin diseases. They are also widely applied to drug safety evaluation, a cosmetic test and biomedical research, so as to explore the molecular mechanism of the skin health and diseases.
Results are shown in
It can be seen from A in
It can be seen from B in
It can be seen from E in
It can be seen from C in
It can be seen from F in
The sample is analyzed by LC-MS/MS equipped with an on-line nano-spray ion source. A total of 3 μL of sample is loaded, and separated in a gradient of 60 min. A column flow rate is controlled at 300 nL/min, a column temperature is 40° C., and an electrospray voltage is 2 kV. The gradient starts from a 2% B phase, is increased to 35% in a nonlinear gradient within 47 min, is increased to 100% within 1 min, and is maintained for 12 min.
A mass spectrometer operates in data-dependent acquisition mode, and is automatically switched between MS and MS/MS acquisition. Mass spectrometric parameters are set as follows: (1) MS: scanning range (m/z): 200-2000; resolution: 70,000; AGC target: 3e6; and maximum injection time: 50 ms; and (2) HCD-MS/MS: resolution: 17,500; AGC target: 1e5; maximum injection time: 45 ms; collision energy: 28%; and dynamic exclusion time: 30 s.
Library search parameter settings: a tandem mass spectrum is analyzed by PEAKS Studio version 10.6. PEAKS DB searches a uniprot-Salmo salar or uniprot-Acipenser sinensis database, and sets none enzymolysis. For library search parameters, an allowable error of a fragment ion mass is 0.02 Da, and an allowable error of parent ion mass is 7 ppm. A protein card value contains 1 unique peptide; and a peptide card value is −10lgP≥20.
A database of bioactive peptides and computer virtual screening tools are used to analyze, virtually screen and predict potential bioactive peptides in peptide sequences of the enzymolysis product.
All target polypeptide sequences obtained by the mass spectrometer are used for displaying distribution of the polypeptides in the sample in an Upset Venn diagram.
Peptide Ranker is used to predict and rank the potential biological activities of peptide fragments; and CPPpred is used to predict and rank cell permeability, which can evaluate cell penetration potentials of the peptides. Scores of the above two are between 0 and 1. The higher the score is, the greater the potential is.
A peak area of each peptide represents the content to a certain extent, so the peak area can be used as a filtering condition to improve the accuracy of the identified peptide fragments and screen a main contributor peptide fragment. Therefore, with the Peptide Ranker score>0.5, the CPPpred score>0.1, and a relative peak area>0.05% as the filtering conditions, the potential bioactive peptide is screened out. With the Peptide Ranker score as an x-axis, the CPPpred score as a y-axis, and the peak area of the peptide as an area of a circle, a bubble chart is made to show a relationship among the three.
There is a relationship among a protein function, a charge and hydrophobicity, and a net charge, the hydrophobicity, etc. can be used for describing protein intermolecular force. Researches have found that the hydrophobicity of the proteins has a significant correlation with emulsifying properties, solubility, etc. Therefore, PepDraw is used to measure the chemical properties of the polypeptides: an isoelectric point, the net charge, and the hydrophobicity.
By using mass spectrometry and virtual screening methods, combined with peptide ranker, the peak area, CPPpred prediction and other indexes are used for screening the potential bioactive peptides in the enzymolysis products of the salmon roe.
Results are shown in Table 5 and
It can be seen from Table 5 that in this research, it is obtained that there are 15 polypeptide sequences with the potential biological activity in the enzymolysis product of the salmon roe. Combined with related data such as polypeptide activity scores in
In this research, C57BL/6J mice are selected for an experiment, and divided into 8 groups: a normal group, a model group, a low-dosage sample group, a high-dosage sample group, two pure peptide sample groups, and two control groups, with 5 mice in each group. Except for the normal group, from the beginning of the experiment, the mice in the sample groups and the model group are subcutaneously injected with 5% D-galactose solution (125 mg/kg) in the scruff of the necks every day, to establish an aging model for 8 consecutive weeks. The normal group and the control groups receive injection with the same amount of saline. From the 8th d of modeling, the mice in the sample groups are treated with corresponding intragastric samples for 7 consecutive weeks. The control groups and the model group are continued to receive intragastric administration with an equal volume of normal saline. During the experiment, all the mice are fed with a normal feed in an environment of 20-24° C., and operations are performed according to specific dosage indexes.
Specific dosage information is shown in Table 6.
In order to evaluate oxidative stress related indexes, 3% pentobarbital sodium (30 mg/kg body weight) is used to perform intraperitoneal anesthesia on the mice 1 h after the last administration, and then blood is collected from orbital venous plexuses of the mice. The obtained blood is stored in a standard blood collection tube, and placed at the room temperature for 1.5 h. Subsequently, the blood is centrifuged at 3000 rpm for 10 min, and a supernatant is taken. The supernatant is used to measure the activities of the superoxide dismutase (SOD), the glutathione (GSH), and the glutathione peroxidase (GSH-Px) and the content of the malondialdehyde (MDA) complying with instructions of the kits.
In addition, in order to measure skin-related biochemical indexes, a skin tissue sample is taken from a hair removal area of a back of each mouse, and it is ensured to remove subcutaneous tissues below it, with a sampling amount of about 0.1 g. The tissue sample is rinsed for 2 times in pre-cooled normal saline, and then water on the surface is dried with filter paper. Then, the tissues are placed in a 4 mL EP tube, and cut into small fragments. Under ice bath conditions, pre-cooled normal saline is added to the tissue fragments (with a final tissue concentration of 0.1 g/mL) for homogenization. Then, a resultant is centrifuged at 12000 rpm for 20 min, and a supernatant is taken. Finally, these supernatants are used to measure the contents of the collagen and the hyaluronic acid (HA) complying with steps of the kits.
In this research, SD rats are used, and divided into three groups: a normal group and salmon YLP groups (with a daily dosage of 100 mg/kg bw). The rats are fasted but allowed to drink water 12 h before the experiment begins. At 15 min, 45 min, 90 min, 180 min and 360 min after intragastric administration, all the rats are anesthetized with the 3% pentobarbital sodium, and blood is collected from abdominal aortas, followed by euthanasia. A polypeptide sample is prepared by solid phase synthesis, and desalted.
(1) The required samples are taken out for thawing, and uniformly mixed by a vortex.
(2) 40 μL of a mixed sample is taken, and 120 μL of methanol is added for shaking treatment for 10 min, and a resultant is centrifuged at a low temperature for 10 min.
(3) 70 μL of a supernatant solution is taken from the centrifuged sample for subsequent measurement.
Concentrations of standards are shown in Table 7.
Chromatographic separation is performed by using a Waters ACQUITY UPLC I-CLASS ultra-high performance liquid chromatographic device. Specific conditions are as follows: chromatographic column: Waters UPLC HSS T3 (1.8 μm, 2.1 mm×100 mm)-mobile phase: A phase (water, 0.1% formic acid), B phase (methanol)-flow rate: 0.3 mL/min-injection volume: 5.0 μL-column temperature: 40° C.-single sample analysis time: 8 min.
A gradient program of the mobile phases is shown in Table 8.
A Waters XEVO TQ-XS tandem quadrupole mass spectrometer is used for mass spectrometry detection. Specific conditions are as follows: positive ion source voltage: 3.0 kV-cone voltage: 30 V-desolvation temperature: 500° C.-desolvation gas flow rate: 1000 L/h-cone gas flow rate: 150 L/h.
Data is expressed in the form of Mean±SD. Graphpad is used for data processing and significance analysis. P<0.05 and P<0.01 indicate significant and very significant differences in data. A target peak area in polypeptide metabolic data is calculated by using TargetLynx software, and an allowable error of a retention time is 15 s. Concentration calculation is based on a standard curve method to obtain quantitative results.
Results are shown in
It can be seen from Ain
It can be seen from B in
Results are shown in
Antioxidant indexes can show a body's overall ability to resist aging. It can be seen from A in
The glutathione (GSH) is a recognized antioxidant index. It can be seen from B in
The MDA is a product of lipid peroxidation. An increase in the content of the MDA indicates an increase in lipid oxidation in vivo. It can be seen from C in
The SOD is an important index of an antioxidant defense system in vivo. It can be seen from D in
In conclusion, the enzymolysis product of the salmon can significantly strengthen the antioxidant system of the aging mice, and shows a dosage-dependent limit. The YLP pure peptides have a good effect on improving the oxidative stress states in the aging mice.
Results are shown in
A curve of a corresponding standard is shown in
It can be seen from
In vivo absorption of the salmon roe tripeptide YLP is shown in
It can be seen from
The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. A variety of modifications on these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention cannot be limited to these embodiments shown herein, but conforms to the widest range consistent with the principle and novel features disclosed herein.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202311694473.1 | Dec 2023 | CN | national |
