A plant polypeptide and its application in preparing cataract-inhibiting drug

Abstract

The present invention discloses a plant polypeptide and its application in preparing a drug for inhibiting cataracts, and relates to the field of protein polypeptides. The present invention provides a plant polypeptide for treating cataracts, which comprises a peptide segment with an amino acid sequence of ARIEAMDSFNGRA. The present invention successfully separates the plant polypeptide ARIEAMDSFNGRA from soybean protein powder. The active peptide can promote the activity of lens epithelial cells, has good effects in preventing and treating cataracts, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the field of protein polypeptides, and in particular to a plant polypeptide and application thereof in the preparation of a drug for inhibiting cataracts. Background Art

[0002] Aging, radiation, trauma, heredity, poisoning, immunity, local metabolism and nutritional disorders, etc., a variety of congenital or acquired factors can affect the metabolism of the lens, causing the denaturation of lens proteins, resulting in lens opacity, which is called cataract. Since the cloudy lens will block the light from entering the eye, cataract patients generally have poor vision or even blindness. Among many eye diseases, cataract is the leading cause of blindness in China and even in the world. Among the 40-45 million blind people in the world, 46% are blinded by cataracts. Cataracts are more common in the elderly. As people age, the incidence of cataracts increases significantly. The pathogenesis of cataracts is complex and is related to multiple factors such as aging, metabolism, physical and chemical damage and heredity. It is the result of the long-term combined action of multiple causes inside and outside the body. Although the specific mechanism is not clear, it is recognized by domestic and foreign studies that oxidative stress and free radical damage are common pathways for various factors to cause cataracts. Anti-aging actives may be effective in preventing the development of cataracts, reversing cataracts in a patient, reducing the severity of cataracts, or improving lens clarity.

[0003] Soybeans are the hydrolyzed products of soy protein. So far, studies have shown that soy peptides obtained by hydrolysis of soy protein have a small molecular weight, are rich in a variety of bioactive peptides, and have many potential health benefits, including good DPPH free radical scavenging activity and in vitro reducing ability, lowering blood pressure, regulating immunity and anti-fatigue, etc. The development and use of soy peptides with high safety and antioxidant activity has always been a hot topic in research.

[0004] Therefore, technicians in this field are committed to developing bioactive peptides that can repair oxidative stress damage from soybeans, reverse the oxidative damage of cataracts, and improve lens clarity. Summary of the invention

[0005] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is to address the current situation that there are relatively few preparations and drugs for treating cataracts.

[0006] To achieve the above object, the present invention provides a plant polypeptide for treating cataract, comprising a polypeptide.

[0007] Preferably, the amino acid sequence of the polypeptide is ARI EAMDSFNGRA.

[0008] Preferably, the polypeptide is derived from soybean.

[0009] In a preferred embodiment of the present invention, the use of the polypeptide in the preparation of a drug for preventing and treating cataracts

[0010] Preferably, the polypeptide is prepared by recombinant expression and chemical synthesis.

[0011] In another preferred embodiment of the present invention, the present invention provides a drug comprising the above polypeptide.

[0012] Preferably, the drug further comprises sodium hyaluronate, boric acid, disodium edetate and ethyl parahydroxybenzoate.

[0013] Preferably, the medicine is an injection, tablet, capsule, oral solution, granule, ointment, suppository and aerosol.

[0014] Preferably, the drug is eye drops, eye drops, cleaning solutions, eye ointments, and intravitreal injections.

[0015] The present invention successfully separates the plant polypeptide ARI EAMDSFNGRA from soybean protein powder. The active peptide can promote the activity of lens epithelial cells, has good effects in preventing and treating cataracts, and has broad application prospects.

[0016] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is the elution curve diagram on the gel column after enzymatic hydrolysis;

[0018] Figure 2 This is the result diagram of the protective efficacy of each component after Sephadex G-15 chromatography on HLE-B3 cells induced by H2O2 oxidative damage;

[0019] Figure 3 This is a graph showing the protective effect of peptide 1-7 on HLE-B3 cells induced by H2O2 oxidative damage;

[0020] Figure 4 The graph shows the results of the proliferation activity of peptide 6 at different concentrations on a high glucose-induced cataract cell model. DETAILED DESCRIPTION

[0021] The following describes several preferred embodiments of the present invention with reference to the drawings in the specification, so that the technical content is clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned in the text.

[0022] Example 1

[0023] Soy protein powder was prepared into a solution with a solid-liquid ratio of 1:12 w / v (g / mL), pretreated in a water bath at 65°C for 30 min, and then used;

[0024] After the soy protein powder is prepared, the solution is cooled to 40°C, the pH is adjusted to 7.4, Alcalase alkaline protease (3000U / g soy protein powder) is added, and enzymolysis is performed at 40°C and an ultrasonic frequency of 15kHz for 40 minutes;

[0025] After the enzymatic hydrolysis is completed, the enzymatic hydrolyzate is heated to 90°C to inactivate the enzyme, and centrifuged (6500 r / min, 10 min) to obtain the supernatant;

[0026] The supernatant was ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 3 kDa, and the ultrafiltrate less than 3 kDa (containing protein peptides) was further separated and purified using a Sephadex G-15 gel column to obtain component 1, component 2, component 3 and component 4. The sample volume was 250 mL, and the mixture was eluted with distilled water at a flow rate of 2 mL / min, and 5 mL was collected in each tube. Figure 1 As described above, component 1 is obtained by combining tubes 5-13, component 2 is obtained by combining tubes 16-21, component 3 is obtained by combining tubes 29-37, and component 4 is obtained by combining tubes 45-49.

[0027] HLE-B3 cells were purchased and cultured in an incubator (37° C., 5% CO 2 ) for later use.

[0028] To detect the protective effect against oxidative damage of HLE-B3 cells, a ROS kit was used to detect the ROS content in the control group (DMEM complete medium), model group (DMEM complete medium + H2O2), component 1 sample group (containing DMEM complete medium + H2O2), component 2 sample group (containing DMEM complete medium + H2O2), component 3 sample group (containing DMEM complete medium + H2O2) and component 4 sample group (containing DMEM complete medium + H2O2). 10 μM DCFH-DA fluorescent probe was added to each experimental group, and the unloaded fluorescent probe was washed with PBS after incubation in the incubator for 20 min. The cells were collected and the fluorescence intensity was detected by flow cytometry.

[0029] like Figure 2 As shown, the component 2 sample group can significantly inhibit ROS in HLE-B3 cells and significantly reduce hydrogen peroxide-mediated oxidative stress.

[0030] Example 2

[0031] The component 2 sample group was subjected to composition analysis and amino acid sequence determination using LC-ES IQ-TOF-MS / MS technology. The m / z of the electrospray ionization (ES I) mass spectrometer was set to 100-1500 positive ion mode, the ionization baffle voltage was set to 500 V, the capillary voltage was set to 4500 V, the nebulizer pressure was set to 1 bar, and the quadrupole ion energy and collision-induced dissociation energy were set to 5.0 eV and 10.0 eV, respectively. Mobile phase A: water (containing 0.3% formic acid); mobile phase B: acetonitrile (containing 0.3% formic acid).

[0032] The collected LC-ES IQ-TOF-MS / MS data were analyzed using Mascot Directory software and compared with databases such as NCB I nr to obtain the amino acid sequence of short peptides with biological activity, as shown in Table 1.

[0033] Table 1 Amino acid sequences of short peptides with biological activity

[0034]

[0035]

[0036] Example 3

[0037] Peptides 1-7 in Example 2 were prepared by organic solid phase synthesis, and the HLE-B3 oxidative damage model in Example 1 was used to detect the protective efficacy of peptides 1-7 in Example 2 against oxidative damage.

[0038] like Figure 3 As shown, peptide 6 (ARI EAMDSFNGRA) has a very significant inhibitory effect on oxidative damage of lens epithelial cells, and peptide 6 can be used as a candidate drug for the treatment of cataracts.

[0039] Example 4

[0040] Excessive apoptosis of lens epithelial cells has always been considered as one of the main pathogenic mechanisms of cataracts. Diabetes causes many eye diseases, among which cataracts are one of the main causes of vision loss in diabetic patients. A cataract cell disease model can be constructed by treating HLE-B3 cells with high sugar. HLE-B3 cells are constructed into the following experimental groups: control group, high sugar group (model group), high sugar + low concentration short peptide, high sugar + medium concentration short peptide and high sugar + high concentration short peptide, among which the concentration of low concentration short peptide is 0.02 mg / mL, the concentration of medium concentration short peptide is 0.05 mg / mL, and the concentration of high concentration short peptide is 0.1 mg / mL.

[0041] After culturing for 24 hours, CCK-8 was used to detect the cell activity of each experimental group. 10 μl of CCK-8 was added to each well and cultured in a 37°C CO2 incubator for 2 hours. The OD value at 450 nm was measured on an ELISA reader.

[0042] like Figure 4 As shown, compared with the blank control group, the viability of lens epithelial cells in the high glucose group was significantly reduced, while in the high glucose group with the addition of peptide 6, as the concentration of peptide 6 increased, it had a significant promoting effect on the proliferation activity of lens epithelial cells under high glucose conditions.

[0043] Example 5

[0044] Three-week-old rats with no ocular abnormalities were selected and divided into a control group, a model group, a peptide 6 0.1 group (with a short peptide concentration of 0.1 mg / mL) and a model + peptide 6 0.1 group (with a short peptide concentration of 0.1 mg / mL). Compound tropicamide eye drops were used to dilate the pupils of the model group, the peptide 6 0.1 group and the model + peptide 6 0.1 group. When disc-like opacities accompanied by vacuoles appeared in the anterior and / or posterior subcapsular cortex of the rat lens, modeling was stopped. The model group and the model + peptide 6 0.1 group continued to be injected with streptozotocin. After 20 days, the degree of lens opacity in the control group, the model group, the peptide 6 0.1 group and the model + peptide 6 0.1 group was observed.

[0045] Lens opacity classification:

[0046] I: Lens Clarification

[0047] II: fine punctate opacities appear in the subcapsular cortex of the lens and are arranged in a ring shape, accompanied by vacuoles;

[0048] III: discoid opacities with vacuoles in the anterior and / or posterior subcapsular cortex of the lens;

[0049] Stage III: The cortex under the posterior lens capsule shows honeycomb opacity, which gradually becomes thinner towards the equator and is accompanied by vacuoles.

[0050] Stage IV: The lens is completely opacified.

[0051] After 20 days, the degree of lens opacity in the control group was I, and the model group had become completely opaque under the action of streptozotocin. In the peptide 6 0.1 group, the fine dot-like opacities in the subcapsular cortex of the lens were alleviated, and the number of vacuoles was significantly reduced. In the model + peptide 6 0.1 group, the opacity of the posterior subcapsular cortex was further aggravated, and the number of vacuoles increased, but the degree of lens opacity was not as severe as that of the model group. It can be seen that 0.1 mg / mL peptide 6 has a therapeutic and preventive effect on cataracts.

[0052] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A plant polypeptide for treating cataracts, characterized in that: The amino acid sequence of the polypeptide is ARIEAMDSFNGRA.

2. The plant polypeptide according to claim 1, characterized in that The polypeptide is derived from soybean.

3. A drug containing the polypeptide according to any one of claims 1-2.

4. The drug according to claim 3, characterized in that The drug also contains sodium hyaluronate, boric acid, disodium edetate and ethyl paraben.

5. The drug according to claim 3, characterized in that The polypeptide is prepared by recombinant expression and chemical synthesis.

6. The drug according to claim 3, characterized in that The medicine is in the form of injection, tablet, capsule, oral liquid, granule, ointment, suppository or aerosol.

7. The drug according to claim 3, characterized in that The medicine is eye drops.

8. The drug according to claim 7, characterized in that The eye drops are eye drops, cleaning solutions, eye ointments or intravitreal injections.

9. Use of the plant polypeptide according to any one of claims 1 to 2 or the drug according to any one of claims 3 to 8 in the preparation of a drug for treating cataracts.

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