Use of bioactive substances in preparing compositions for relieving eye fatigue caused by blue light and / or preventing eye inflammation caused by blue light

Abstract

Use of a bioactive substance for the preparation of an ophthalmic health and / or anti-inflammatory composition, wherein the bioactive substance is a peptide having an amino acid sequence as set forth in SEQ ID NO: 1, wherein the amino acid sequence is a fragment of a bovine skin peptide; wherein the composition is used to reduce the expression level of at least one of an inflammation-related gene comprising a vascular endothelial growth factor A gene, an interleukin-1β gene, and an interleukin-8 gene.

Description

TECHNICAL FIELD

[0001] The present application relates to the use of a bioactive substance, in particular to the use of a peptide as a bioactive substance for preparing an eye care and / or anti-inflammatory composition. BACKGROUND

[0002] With the rapid development of technology products, modern people spend a lot of time using mobile phones and computers, which gradually increases the burden on the eyes, and is prone to visual function decline and dry eye syndrome and other problems, so eye care is a very important issue in today's society. In addition, the damage caused by blue light to the eyes cannot be ignored, and can even cause eye inflammation and other symptoms.

[0003] The clinical symptoms of eye inflammation include red eyes, itchy eyes, conjunctival edema, eyelid swelling, and tearing, etc. It is caused by external stimulation to activate the vascular endothelial cell receptor, leading to vasodilation and increased vascular permeability, thereby promoting inflammatory factors such as IL-1β and IL-8 into the blood vessels, promoting white blood cells into the conjunctival tissue, and ultimately causing inflammation. Common diseases such as allergic conjunctivitis, etc.

[0004] Collagen is an important protein in the human body, which exists widely in connective tissue. It can lubricate joints and keep the skin elastic. With the loss of collagen, the skin will produce wrinkles and relaxation and other phenomena. In addition to being the main component of human ligaments and extracellular matrix and other tissues, collagen is also the main component of the cornea of the eye.

[0005] There is an urgent need for an effective eye care nutrition product in today's society, and there is no better research to explore the eye care efficacy of collagen-related peptides. In addition, whether specific collagen-related peptide fragments have eye care or anti-inflammatory efficacy needs to be clarified. SUMMARY

[0006] Therefore, the purpose of the present application is to provide the use of a bioactive substance for preparing an eye care and / or anti-inflammatory composition, wherein the bioactive substance is a peptide, and the peptide has an amino acid sequence as set forth in SEQ ID NO: 1, wherein the amino acid sequence is a bovine leather peptide fragment.

[0007] In some embodiments, the amino acid sequence can be isolated and purified from bovine leather cells, bovine leather collagen, or bovine meat cells.

[0008] In some embodiments, the amino acid sequence can be synthesized by solid phase synthesis (Fmoc-Solid Phase Peptide Synthesis) or other chemical synthesis methods.

[0009] In some embodiments, wherein the composition is used to reduce the expression of inflammation-related genes, the inflammation-related genes comprise at least one of a vascular endothelial growth factor A (VEGFA) gene, an interleukin-1 beta (IL-1β) gene, and an interleukin-8 (IL-8) gene.

[0010] In some embodiments, wherein the concentration of the bioactive substance is at least 0.0125 mg / mL.

[0011] In some embodiments, wherein the eye care comprises preventing eye inflammation, anti-blue light, relieving eye fatigue, and reducing light damage to the eye.

[0012] In some embodiments, wherein the composition is a pharmaceutical product, a food product, or a care product.

[0013] In some embodiments, wherein the food product is a health food product.

[0014] In summary, the present application provides a use of a bioactive substance for preparing an eye care and / or anti-inflammatory composition, wherein the bioactive substance is a peptide having an amino acid sequence as set forth in SEQ ID NO: 1, wherein the amino acid sequence is a peptide fragment of a cowhide; the amino acid sequence has the effect of reducing the expression of inflammation-related genes, the inflammation-related genes comprise at least one of a vascular endothelial growth factor A (VEGFA) gene, an interleukin-1 beta (IL-1β) gene, and an interleukin-8 (IL-8) gene, thereby achieving the effects of preventing eye inflammation, anti-blue light, relieving eye fatigue, reducing light damage to the eye, slowing down visual function decline, preventing dry eye, and anti-inflammation.

[0015] The embodiments of the present application will be further described with reference to the drawings. The following examples are presented to illustrate the features and applications of the present application, but not to limit the scope of the present application. Any person skilled in the art can make some modifications and refinements without departing from the spirit and scope of the present application, and the scope of protection of the present application is defined by the appended claims. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is a graph of the relative ratio of the expression of the VEGFA gene of each group in Example 3;

[0017] Figure 2 is a graph of the relative ratio results for IL-1 β gene expression for each group in Example Three;

[0018] Figure 3 is a graph of the relative ratio results for IL-8 gene expression for each group in Example Three. DETAILED DESCRIPTION

[0019] Some embodiments of the present application will be further described by way of illustration with reference to the accompanying drawings. These examples are exemplary and are not intended to limit the scope of the present application. Any modification and refinement made by those skilled in the art without departing from the spirit and scope of the present application should not be excluded from the scope of protection.

[0020] Statistical analysis was performed using Excel software. Data were expressed as mean ± standard deviation (SD), and differences between groups were analyzed by student’s t-test. In the figures, “*” represents p value less than 0.05, “**” represents p value less than 0.01, and “***” represents p value less than 0.001. The more “*”, the more significant the statistical difference.

[0021] In some embodiments, any of the aforementioned compositions can be a consumable composition. In other words, a consumable composition comprises a specific amount of the bioactive substance. In some embodiments, the aforementioned consumable composition can be a food product or a food additive. In some embodiments, the food product can be, but is not limited to, beverages, fermented foods, bakery products, health foods, or dietary supplements.

[0022] In some embodiments, the use of a peptide as a bioactive substance for the preparation of an eye care and / or anti-inflammatory composition, wherein the bioactive substance is a peptide having an amino acid sequence as set forth in SEQ ID NO: 1, wherein the amino acid sequence is a peptide fragment of a bovine skin.

[0023] It should be understood that the "peptide" is a substance between amino acids and proteins, and is composed of a plurality of amino acids. Also, the peptide as a bioactive substance can be an "isolated peptide" or a "synthetic peptide". Among them, the "isolated peptide" refers to a peptide fragment isolated from an organism or an organism derivative, and the peptide fragment has a biological activity. The "synthetic peptide" refers to a peptide fragment synthesized according to the desired amino acid sequence by an instrument or artificial experimental operation, and the peptide fragment has a biological activity. Also, the term "isolated peptide" described herein is equivalent to "isolated peptide" or "isolated peptide", and the term "synthetic peptide" is equivalent to "synthetic peptide" or "synthetic peptide".

[0024] It should be understood that the term "protein" described herein is equivalent to "protein", for example, the term "collagen protein" is equivalent to "collagen protein".

[0025] In some embodiments, the peptide as a bioactive substance can be isolated from the peptide fragment of the cowhide or synthesized by an instrument or artificial experiment. For example, the source of the peptide fragment of the cowhide includes cowhide cells, collagen (hereinafter referred to as cowhide collagen), and beef cells. Since the main component of the cowhide is collagen, and in the process of extracting the cowhide collagen from the cowhide, in addition to the main extracted cowhide collagen, the proteins in the cowhide cells (i.e., cowhide cell proteins) and the proteins in the beef cells remaining on the cowhide (i.e., beef cell proteins) are also included. It should be understood that the term "peptide fragment of the cowhide" described herein refers to a peptide fragment mainly composed of collagen, and a peptide fragment containing cowhide cell proteins and beef cell proteins.

[0026] In some embodiments, the peptide fragment of the cowhide can include at least one collagen, procollagen, cowhide cell protein, beef cell protein, or a combination thereof. For example, the collagen can be collagen type IV, procollagen type V, collagen type XIV, etc.

[0027] For example, the collagen peptide raw material of the cowhide can be separated to obtain the amino acid sequence shown in SEQ ID NO: 1, and the collagen peptide raw material includes the peptide fragment of the cowhide collagen, the peptide fragment of the cowhide cell protein, and / or the peptide fragment of the beef cell protein. In an embodiment, the collagen peptide raw material can be German cowhide collagen peptide powder (purchased from GELITA, Germany), or the collagen peptide powder formed by enzymatic hydrolysis and drying of the collagen extracted from the cowhide.

[0028] In some embodiments, the bioactive peptide can be isolated from bovine collagen peptide powder using instruments (e.g., fast protein liquid chromatography and high-performance liquid chromatography systems). Furthermore, the isolation step utilizes the peptide's physical or chemical properties (e.g., molecular weight, hydrophilicity, polarity, non-polarity, etc.) to isolate the amino acid sequence of SEQ ID NO: 1.

[0029] In some embodiments, the amino acid sequence can be synthesized by solid phase synthesis (Fmoc-Solid Phase Peptide Synthesis) or other chemical synthesis methods.

[0030] In some embodiments, the composition prepared from the amino acid sequence of SEQ ID NO: 1 is used to reduce the expression of inflammation-related genes, wherein the inflammation-related genes include at least one of the vascular endothelial growth factor A (VEGFA) gene, the interleukin-1β (IL-1β) gene, and the interleukin-8 (IL-8) gene.

[0031] In some embodiments, the concentration of the bioactive substance is at least 0.0125 mg / mL.

[0032] In some embodiments, the bioactive substances described herein are used to prepare eye care and / or anti-inflammatory compositions, wherein the eye care includes preventing eye inflammation, resisting blue light, relieving eye fatigue, and reducing light damage to the eyes.

[0033] In some embodiments, the composition is a medicine, a food, or a skin care product, wherein the food is a health food.

[0034] In some embodiments, the aforementioned composition may be an edible composition. In some embodiments, the edible composition may be prepared as a food product or a food additive, meaning that the composition is added to food ingredients during food preparation using conventional methods or during the production process of the food product. In this context, the food product may be formulated with edible materials for human or animal consumption.

[0035] In some embodiments, the food product may be, but is not limited to, beverages, fermented foods, bakery products, health foods, and dietary supplements.

[0036] Example 1. Peptide isolation of bovine collagen

[0037] First, 100 mg of bovine collagen peptide powder (purchased from GELITA, Germany) was weighed and dissolved in 5 mL of buffer A to obtain a collagen peptide solution. The buffer A was prepared by mixing 50 mM Tris / HCl buffer (pH 8.0) and 100 mM NaCl.

[0038] Next, the collagen peptide solution was subjected to a rough separation using a fast protein liquid chromatography (FPLC purification instrument, model GE Healthcare Life Sciences, hereinafter referred to as a purification instrument) to obtain a primary separation peptide mixture. The separation column installed in the purification instrument was a sephadex G-25 column (2.6 cm x 10 cm, 53 mL). The flow rate of the purification instrument was set to 1 mL / min, and the wavelength of the ultraviolet light used for observation was 220 nm. Furthermore, the primary separation peptide mixture with a peak value of 5 kDa or less was freeze-dried at -80°C for 12 hours using an instrument (EYELA; model: FD-1000) to obtain a solid primary separation peptide mixture.

[0039] Next, the pre-separation peptide mixture was separated using a high-performance liquid chromatography (HPLC) system (model Hitachi Chromaster HPLC system, Hitachi, Tokyo, Japan), hereinafter referred to as an HPLC system, to obtain a plurality of secondary separation peptides. The HPLC system was equipped with a molecular sieve C18 high-pressure column (model TSKgel G2000SWXL, Tosoh, 30 cm x 7.8 mm, 5 μm). In the set values of the HPLC system, buffer solution A (0.1% TFA dissolved in 100% deionized water) and buffer solution B (0.1% TFA dissolved in 100% ACN) were mixed according to the separation gradient, and the separation gradient was 5% acetonitrile (ACN) / 0.1% TFA to 100% acetonitrile / 0.1% TFA (i.e., the concentration of ACN was gradiently increased from 5% to 100% in a 0.1% TFA solution environment), the flow rate was set to 1 mL / min, and the column temperature was set to 40°C.

[0040] Here, the peptides in the initial peptide mixture are eluted from the HPLC solution with different polarities and molecular weights, and then a plurality of separated peptides are obtained. Further, the plurality of separated peptides are freeze-dried at -80°C for 12 hours (instrument model: EYELA; model number: FD-1000) to obtain a plurality of solid separated peptides.

[0041] Example 2. Peptide identification of bovine collagen

[0042] The plurality of separated peptides of Example 1 were subjected to protein identification. First, the plurality of solid separated peptides were prepared at a concentration of 20 mg / ml with deionized water, and then subjected to protein identification using a liquid chromatography mass spectrometer (LC-MS / MS). Further, the liquid chromatography mass spectrometer (LC-MS / MS) was a quadrupole-time-of-flight tandem mass spectrometer system (Q-TOF), in which the liquid chromatography system (LC system) was an UltiMate 3000 RSLCnano LC Systems (model: Thermo Fisher Scientific), and the mass spectrometer (Mass Spectrometer) was a 6600 System (model: Applied Biosystems Sciex).

[0043] The separation column installed in the liquid chromatography system was a C18 separation column (Acclaim PepMap C18, 75 μm I.D. x 25 cm nanoViper, 2 μm, 300 A, Thermo Fisher Scientific). The solution system used in the liquid chromatography mass spectrometer was buffer solution A (0.1% TFA in 100% deionized water) and buffer solution B (0.1% TFA in 100% ACN). The separation gradient set in the liquid chromatography mass spectrometer was 5% buffer solution B to a gradient to 90% buffer solution B, the flow rate was set to 300 nanoliters per minute (300 nl / min), and the gradient time was 30 minutes.

[0044] In the setting values of the mass spectrometer, the survey scan was set to scan all ionized separated peptides in the range of 400 m / z (mass-to-charge ratio) to 1200 m / z. In the information-dependent acquisition (CID) mode, the detection range of the peptides was set to 100-5000 dalton (Da).

[0045] ​​Peptides are particularly susceptible to fragmentation at the peptide bond in the gas phase environment of mass spectrometry, a, b, c series ions are N-terminal fragments, x, y, z are C-terminal fragments, and the numbers represent the order of amino acids. The entire sequence of the peptide can be calculated from the mass difference between fragments of the same series (such as y1, y2, y3…), wherein the amino acid sequence and molecular weight shown in SEQ ID NO: 1 are identified.

[0046] Table 1.

[0047]

[0048] As can be seen from Table 1, in some embodiments, the molecular weight of the isolated peptide of the present application is 1110.66, and its structure is as follows:

[0049]

[0050] It is known that the bovine collagen peptide raw material contains the amino acid sequence of the isolated peptide described above, i.e., SEQ ID NO: 1.

[0051] Example 3. Synthesis of bovine collagen peptide

[0052] To verify the efficacy of the isolated peptide amino acid sequence identified in Example 2 for eye health, a synthetic peptide was prepared in Example 3 according to the amino acid sequence of the amino acid sequence identified in the aforementioned Example 2 (i.e., SEQ ID NO: 1). The synthesis method used was Fmoc-Solid Phase Peptide Synthesis, and the instrument used was a peptide synthesizer (Model Focus XC III 0, AAPPTEC, USA).

[0053] It is known that the amino acid sequence of SEQ ID NO: 1 is Thr-Lys-Leu-Pro-Ile-Gly-Pro-Ser-Gly-Leu-Pro-Val-Ser-Leu.

[0054] Step 1: First, place the resin in the reaction tube, and according to 15 ml of dichloromethane (DCM) per 1 gram of resin, soak the resin in dichloromethane for 30 minutes to swell the resin in the solution.

[0055] Step 2: Remove the dichloromethane in the reaction tube, and add 15 mL of 20% piperidine dimethylformamide (piperidine DMF) solution per 1 gram of resin to the reaction tube to react with the resin for 5 minutes, then remove the solution in the reaction tube. Again, add 15 mL of 20% piperidine dimethylformamide solution per 1 gram of resin to the reaction tube to react with the resin again for 15 minutes to remove the protecting group on the resin to obtain the deprotected resin.

[0056] Step 3: After removing the solution in the reaction tube again, take out ten particles of the resin from the reaction tube for testing. First, wash the resin with ethanol three times, and add one drop of ninhydrin and phenol solution. Heat at 105°C to 110°C for 5 minutes, and when the ninhydrin and phenol solution and the resin both turn dark blue, it is a positive reaction, indicating that the resin in the reaction tube at this time is deprotected resin and can be combined with amino acids.

[0057] Step 4: Repeat the washing of the deprotected resin by adding 10 mL of dimethylformamide per 1 gram of resin to the reaction tube six times.

[0058] Step 5: Dissolve a three-fold excess of protected threonine (Fmoc-Thr) and a three-fold excess of hydroxybenzotriazole (HOBt) in a small amount of dimethylformamide, and add to the reaction tube containing the deprotected resin to react for 90 minutes.

[0059] Step 6: After 90 minutes of reaction, repeat the washing of the resin combined with amino acids by adding 10 mL of dimethylformamide per 1 gram of resin to the reaction tube three times.

[0060] Then, repeat the above steps (2) to (6) until the remaining amino acids (Lys, Leu, Pro, Ile, Gly, Pro, Ser, Gly, Leu, Pro, Val, Ser, Leu) are sequentially combined to form the primary synthetic peptide with the amino acid sequence of SEQ ID NO: 1.

[0061] Step 7: Repeat the washing of the primary synthetic peptide by adding 10 mL of dimethylformamide per 1 gram of resin to the reaction tube three times, then adding 10 mL of dichloromethane per 1 gram of resin to the reaction tube to wash the primary synthetic peptide three times, and finally adding 10 mL of ethanol per 1 gram of resin to the reaction tube to wash the primary synthetic peptide three times.

[0062] Step 8: React the washed primary synthetic peptide with 10 grams of cleavage solution (86% trifluoroacetic acid, 4% benzyl mercaptide, 3% water, 5% ethanedithiol (EDT), and 2% phenol) for 120 minutes to separate the primary synthetic peptide from the resin.

[0063] Step 9: The cleavage solution containing the primary synthesized peptide was separated from the resin by a sand core funnel, and then the cleavage solution containing the primary synthesized peptide was added to eight times the volume of diethyl ether relative to the cleavage solution to react. Then, the primary synthesized peptide and the cleavage solution were separated by suction filtration with a Buchner funnel, and after the diethyl ether containing the cleavage solution was suctioned dry, the primary synthesized peptide was washed with diethyl ether three times, at which time the primary synthesized peptide was a solid. After the diethyl ether was allowed to evaporate at room temperature, dry primary synthesized peptide was obtained.

[0064] Step 10: 1 mg of the dry primary synthesized peptide was dissolved in 0.5 mL of deionized water, and then 20 mL of the dissolved primary synthesized peptide was separated and purified by an HPLC system (model Hitachi Chromaster HPLC system, brand Hitachi, Tokyo, Japan) to obtain a pure synthesized peptide. In the HPLC system, a C18 column (brand Gemini-NX) was set, and the detection length was set to 220 nm, and in the HPLC system, buffer solution A (0.1% TFA dissolved in 100% deionized water) and buffer solution B (0.1% TFA dissolved in 100% ACN) were mixed according to the linear separation gradient to elute and separate the synthesized peptide. The separation gradient was set to linearly increase from 10% buffer solution B to 90% ACN (dissolved in 0.1% TFA), the flow rate was set to 1 mL / min, and the separation time was set to 30 minutes. According to the peak area of each synthesized peptide calculated from the HPLC chromatogram, the purity of the synthesized peptide was found to be more than 95%. In this way, a synthesized peptide with the amino acid sequence of SEQ ID NO: 1 was obtained.

[0065] Example 4. Efficacy test of eye-protecting and anti-inflammatory genes

[0066] This example uses an RNA extraction kit, reverse transcriptase, KAPA FAST qPCR reagent set in combination with a quantitative PCR instrument to determine the change in the expression amount of eye-protecting and anti-inflammatory genes in human retinal pigment epithelial cells after treatment with the peptide with the amino acid sequence of SEQ ID NO: 1, thereby confirming the efficacy of the bovine peptide of the bioactive substance of the present application against blue light and anti-inflammatory response.

[0067] For example, the inflammation-related genes are vascular endothelial growth factor A (VEGFA) gene, interleukin-1 beta (IL-1β) gene and interleukin-8 (IL-8) gene. The aforementioned inflammation-related genes are genes that are up-regulated when the eye is stimulated by external environment, and are used to promote the generation of inflammatory response, so that the regulation of the aforementioned inflammation-related genes is related to eye health.

[0068] The protein encoded by the vascular endothelial growth factor A (VEGFA) gene is an indispensable component of blood vessel formation in the process of angiogenesis, and is related to the permeability of blood-retinal and blood-brain barriers. The VEGFA protein induces the tyrosin kinase pathway to activate enzymes such as endothelial nitric oxide synthase (eNOS) of endothelial cells, thereby promoting the increase of vascular permeability and angiogenesis. In addition to angiogenesis, VEGFA protein is also involved in the maintenance of the vascular system. The specific binding of VEGFA protein is related to the vascular maturation of various tissues (such as heart, kidney and brain), and VEGFA plays an important role in maintaining static vascular endothelium and promoting inflammatory response.

[0069] The proteins encoded by the interleukin-1 beta (IL-1β) and interleukin-8 (IL-8) genes are important regulatory factors of inflammatory response, which belong to the leukocyte interleukin family proteins. The leukocyte interleukin family includes 11 proteins, which form a complex network of pro-inflammatory response. The leukocyte interleukin family proteins are expressed by leukocytes and endothelial cells in response to external environmental stimuli, thereby initiating and controlling inflammatory response, and are considered as pro-inflammatory response proteins.

[0070] Therefore, in Example 4, VEGFA gene, IL-1β gene and IL-8 gene are used as analysis targets to verify the efficacy of the bioactive substance of the present application on eye protection and anti-inflammatory response.

[0071] Materials and instruments

[0072] 1. Cell strain: human retinal pigment epithelial cells ARPE-19 (purchased from ATCC, product number CRL-2302).

[0073] 2. Culture medium: DMEM medium (Gibco, Cat. No. 11965-092) and Ham's F12 medium (Gibco, Cat. No. 31765035) were mixed at 1:1 volume, and 10% fetal bovine serum (Gibco, Cat. No. 10437-028), 0.5 mM sodium pyruvate (Gibco, Cat. No. 11360-070) and 15 mM HEPES buffer (Gibco, Cat. No. 15630106) were further added.

[0074] 3. RNA extraction kit (Geneaid, Cat. No. Lot No. FC24015-G).

[0075] 4. Reverse transcriptase (III Reverse Transcriptase, Invitrogen, Cat. No. 18080-051).

[0076] 5. Target gene primers for measurement, including VEGFA gene, IL-1β gene, IL-8 gene, and internal control group (GAPDH gene).

[0077] 6. KAPA FAST qPCR reagent kit (Sigma, Cat. No. 38220000000).

[0078] 7. ABI StepOnePlus™ Real-Time PCR system (ABI StepOnePlus™ Real-Time PCR system) from Thermo Fisher Scientific.

[0079] 8. Experimental sample: The experimental sample used in this example is a bioactive substance, specifically a bovine skin peptide, which is a synthetic peptide of SEQ ID NO: 1 (hereinafter referred to as bovine skin peptide) obtained as described in Example 3 above.

[0080] Experimental procedure

[0081] Cell culture stage:

[0082] First, 1.5 x 105human retinal pigment epithelial cells were taken into a six-well cell culture dish containing 2 ml of the above-mentioned culture medium per well, and cultured at 37°C for 24 hours.

[0083] Cell treatment stage:

[0084] ​Next, the human retinal pigment epithelial cells in each well were divided into a control group, a control group (treated with blue light), and an experimental group (added with the peptin of the present application and treated with blue light) according to the following test conditions, with three replicates, and the experimental conditions are shown in Table 2:

[0085] Table 2. Experimental design

[0086] Group Concentration of experimental sample Control of experiment Control group None Incubate for 24 hours Control group None After incubation for 24 hours, irradiate with blue light for 15 minutes Experimental group Bovine epidermis peptide 0.0125 mg / ml After incubation for 24 hours, irradiate with blue light for 15 minutes

[0087] In detail, in the cell treatment stage, the control group and the control group used 2 ml of culture solution to culture the human retinal pigment epithelial cells; the experimental group added the peptin prepared in Example 3 above to the culture solution to make the concentration 0.0125 mg / ml; each group was cultured at 37°C for 24 hours.

[0088] After 24 hours of culture, the control group and the experimental group were irradiated with blue light for 15 minutes.

[0089] Cell analysis stage:

[0090] The human retinal pigment epithelial cells of each group in the cell treatment stage (i.e. the control group, the control group and the experimental group) were broken by cell lysis solution. Next, the RNA in each group of cell solution was extracted by RNA extraction reagent kit (purchased from Geneaid Company, Taiwan, China, Lot No. FC24015-G). Next, 1000 nanograms (ng) of extracted RNA was taken as a template for each group, and the mRNA expression levels of the following genes were detected by RT-PCR: The extracted RNA was reverse-transcribed into corresponding cDNA by III reverse transcriptase (purchased from Invitrogene, USA, product number 18080-051). The cDNA of each group was subjected to quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) by ABI StepOnePlus™ Real-Time PCR system (purchased from Thermo Fisher Scientific), KAPA SYBR FAST (purchased from Sigma, USA, product number 38220000000), and primers of Table 3 (SEQ ID NO: 2 to SEQ ID NO: 9) to observe the expression levels of VEGFA gene, IL-8 gene, and IL-1β gene in human retinal pigment epithelial cells of each group. The instrument setting condition of qRT-PCR was 95°C for 20 seconds, followed by 95°C for 3 seconds, 60°C for 30 seconds, and repeated for 40 cycles, and the gene quantification was performed by using 2-ΔCt method. Here, the qRT-PCR performed by using cDNA can quantify the mRNA expression levels of VEGFA gene, IL-8 gene, and IL-1β gene, and further infer the expression levels of proteins encoded by VEGFA gene, IL-8 gene, and IL-1β gene.

[0091] Table 3

[0092]

[0093]

[0094] *R is REVERSE, and F is FORWARD.

[0095] Please refer to Figure 1 When the expression level of VEGFA gene of the control group was taken as 100%, the expression level of VEGFA gene of the control group relative to the control group was 109.47%, which confirmed that the expression level of VEGFA gene of human retinal pigment epithelial cells would be up-regulated after blue light irradiation, and further promote the production of inflammatory response; and the expression level of VEGFA gene of the experimental group relative to the control group was 84.07%, which, compared with the result of the control group, showed that the expression level of VEGFA gene of the experimental group could be inhibited after the treatment of the experimental group with the peptide of Bovine Luteinizing Hormone Releasing Hormone under the stimulation of blue light.

[0096] Vascular endothelial growth factor A (VEGFA) plays an important role in both physiological and pathological angiogenesis in the eye. When the eye is subjected to external stimuli, it will promote the expression of VEGFA, which in turn leads to inflammation of the eye. Certain molecules have become the target of drug development and have been approved by the US Food and Drug Administration. Bevacizumab is a VEGFA antibody approved for intravenous injection for the treatment of advanced cancer, which has been widely used in the field of ophthalmology for the treatment of exudative age-related macular degeneration, diabetic retinopathy, retinal vein occlusions, retinopathy of prematurity and other chorioretinal vascular disorders. Other VEGFA inhibitors such as Pegaptanib and Ranibizumab are also developed specifically for intraocular use, and their safety and effectiveness in humans have been proven.

[0097] See Figure 2 When the expression amount of IL-1β gene of the control group was taken as 100%, the expression amount of IL-1β gene of the control group relative to the control group was 109.21%, which confirmed that the expression amount of IL-1β gene of human retinal pigment epithelial cells would be up-regulated after blue light irradiation, thereby promoting the production of inflammatory response; and the expression amount of IL-1β gene of the experimental group relative to the control group was 66.36%, compared with the results of the control group, which showed that the expression amount of IL-1β gene of the experimental group could be inhibited after treatment with Bovine Peptide under the stimulation of blue light.

[0098] See Figure 3 When the expression amount of IL-8 gene of the control group was taken as 100%, the expression amount of IL-8 gene of the control group relative to the control group was 121.77%, which confirmed that the expression amount of IL-8 gene of human retinal pigment epithelial cells would be up-regulated after blue light irradiation, thereby promoting the production of inflammatory response; and the expression amount of IL-8 gene of the experimental group relative to the control group was 88.29%, compared with the results of the control group, which showed that the expression amount of IL-8 gene of the experimental group could be inhibited after treatment with Bovine Peptide under the stimulation of blue light.

[0099] IL-1β is a key pro-inflammatory cytokine in the interleukin family of proteins. Upon activation by Toll-like receptors or RIG-like receptors, innate immune cells synthesize IL-1β precursors, which are then modified by proteases to become active IL-1β, thereby promoting inflammatory responses. IL-8 is a cytokine secreted by macrophages and epithelial cells. Interleukin-8 binds to the chemokine receptors interleukin-8 receptor α (IL8RA, also known as CXCR1) and interleukin-8 receptor β (IL8RB, also known as CXCR2), acting as a chemoattractant for neutrophils and regulating inflammatory responses. Interleukin-8 has potent angiogenic effects and plays a key role in inflammatory pathologies such as bronchiolitis and cystic fibrosis. Cytokines / chemokines such as IL-1β and IL-8 have been shown to be involved in the inflammatory mechanisms of eye irritation. When the human body is exposed to eye irritation caused by environmental exposure, the expression levels of IL-1β and IL-8 proteins will increase, causing an inflammatory response in the eye, leading to symptoms such as eyelid swelling, red eyes, and allergic conjunctivitis.

[0100] Figures 1 to 3 The results showed that after blue light stimulation, the expression levels of VEGFA gene, IL-1β gene and IL-8 gene in the control group that was not treated with the bovine peptide of the present invention all showed a downward trend, which promoted the progress of the inflammatory response; however, the expression levels of VEGFA gene, IL-1β gene and IL-8 gene in the experimental group after blue light treatment were all downregulated compared with the control group, indicating that the bovine peptide of the present invention, as a bioactive substance, has the effect of resisting blue light and can reduce the expression levels of genes related to eye inflammation, confirming that the bovine peptide of the present invention does have the actual effect of eye health care and anti-inflammatory response, and can achieve the effects of resisting blue light, relieving eye fatigue, preventing eye inflammation and reducing light damage to the eyes.

[0101] In summary, the peptide as a bioactive substance according to any embodiment of the present application, which includes the amino acid sequence shown in SEQ ID NO: 1, can be prepared for the use of an eye care and / or anti-inflammatory composition. In some embodiments, the amino acid sequence can be isolated and purified from bovine skin cells, bovine skin collagen, or bovine meat cells. In some embodiments, the peptide as a bioactive substance can reduce the expression of inflammation-related genes, including at least one of the vascular endothelial growth factor A (VEGFA) gene, the interleukin-1 beta (IL-1β) gene, and the interleukin-8 (IL-8) gene. Moreover, the prepared composition has an eye care effect, including preventing eye inflammation, resisting blue light, relieving eye fatigue, and reducing light damage to the eyes.

[0102] The technical content of the present application is disclosed in the above-mentioned preferred embodiments, but it is not intended to limit the present application. Any person skilled in the art can make some changes and modifications without departing from the spirit of the present application, and these changes and modifications should be included in the scope of the present application. Therefore, the scope of protection of the present application should be defined by the appended claims.

[0103] Of course, the present application can have other various embodiments. Those skilled in the art can make various corresponding changes and modifications according to the present application without departing from the spirit and essence of the present application, and these corresponding changes and modifications should be included in the protection scope of the claims of the present application. SEQUENCE LISTING <110> Da-Jiang Bio-Medical Technology Co., Ltd. <120> Use of a bioactive substance for the preparation of a composition for relieving eye fatigue caused by blue light and / or preventing eye inflammation caused by blue light <130> N / A <160> 9 <170> PatentIn version 3.5 <210> 1 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 1 Thr Lys Leu Pro Ser Gly Leu Pro Val Ser Leu 1 5 10 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 2 ccttgctgct ctacctccac 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 3 atctgcatgg tgatgttgga 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 4 agctacgaat ctccgaccac 20 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 5 cgttatccca tgtgtcgaag aa 22 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 6 ttttgccaag gagtgctaaa ga 22 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 7 aaccctctgc acccagtttt c 21 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 8 ctgggctaca ctgagcacc 19 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 9 aagtggtcgt tgagggcaat g 21

Claims

1. A use of a bioactive substance for preparing a composition for relieving eye fatigue caused by blue light, characterized in that: The biologically active substance is the amino acid sequence as shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that The amino acid sequence can be isolated and purified from bovine skin cells, bovine skin collagen or beef cells.

3. The use according to claim 1, characterized in that The amino acid sequence can be synthesized by solid phase synthesis or other chemical synthesis methods.

4. The use according to claim 1, characterized in that The composition is used to reduce the expression level of inflammation-related genes, wherein the inflammation-related genes include at least one gene among vascular endothelial growth factor A gene, interleukin-1β gene and interleukin-8 gene.

5. The use according to claim 1, characterized in that The concentration of the biologically active substance is at least 0.0125 mg / mL.

6. Use of a bioactive substance for preparing a composition for preventing eye inflammation caused by blue light, characterized in that: The biologically active substance is the amino acid sequence as shown in SEQ ID NO:

1.

7. The use according to claim 6, characterized in that The amino acid sequence can be isolated and purified from bovine skin cells, bovine skin collagen or beef cells.

8. The use according to claim 6, characterized in that The concentration of the biologically active substance is at least 0.0125 mg / mL.

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