Periocular secretion inhibition gel containing natural plant polyphenol and preparation method thereof

Abstract

The invention provides periorbital secretion inhibition gel containing natural plant polyphenols and a preparation method thereof, and relates to the field of pet eye care, the gel is chelated with iron ions through EGCG to block a porphyrin oxidation pathway, cooperates with zinc ions to inhibit bacterial growth and plays a role in multiple mechanisms of targeted removal of bacterial biofilms through antibacterial peptide LL-37; in-vitro tests prove that the functional components in the gel have a remarkable synergistic effect, proliferation of staphylococcus aureus can be effectively inhibited, deposition of porphyrin is remarkably inhibited, and formation of tear stains is relieved; animal experiment results show that the gel has a remarkable tear stain removal effect and good periorbital safety, after two weeks of treatment, the periorbital tear stain area is reduced by 75.97% compared with that of a bear dog, the eye McDonald-Shiadduck stimulation score is also remarkably reduced, corneal fluorescein staining detection results of all tested animals are negative, and the gel has a good clinical application prospect. A mild and effective new thought is provided for inhibiting the periocular secretions of the pets.

Description

Technical Field

[0001] The present invention relates to the field of pet eye care, and in particular to a periocular secretion inhibiting gel containing natural plant polyphenols and a preparation method thereof. Background Art

[0002] Periocular secretions (such as tear stains and mucus) are common physiological or pathological phenomena in pets, typically formed by a mixture of tears, sebum, dust, microorganisms, and exfoliated cells. Under normal circumstances, small amounts of secretions can be removed through the pet's own cleaning behavior or daily care by the owner. However, if secretions are excessive or accumulate over a long period of time, the porphyrins contained in them will oxidize and become rust-colored, forming tear stains. Short-nosed dog breeds (such as Bichon Frise, Poodle, and Shih Tzu) are particularly prone to persistent tear stains due to abnormal tear duct structure and poor tear drainage. Tear stains not only affect appearance but can also cause periocular skin inflammation, bacterial infection, and even lead to eye diseases such as conjunctivitis.

[0003] Currently, commercially available pet eye cleansing products primarily contain synthetic surfactants, antimicrobials, and bleaching ingredients such as hydrogen peroxide. While these products offer short-term depigmentation, they are highly irritating and can easily cause corneal damage and adverse eye reactions. Long-term use can disrupt the periocular microenvironment, leading to skin dryness, irritation, or allergic reactions. Furthermore, existing products are mostly liquid or wipe formulations. Liquids can easily enter the eye and cause irritation, while wipes can damage delicate skin due to mechanical friction.

[0004] Plant polyphenols are a class of polyhydroxyphenolic compounds naturally found in plants. They have broad-spectrum antibacterial, anti-inflammatory, and antioxidant activities, and due to their low toxicity and biocompatibility, they show potential in the field of pet care. However, existing natural ingredient products for periocular secretions still have the following shortcomings: (1) The effect of a single plant ingredient is limited, making it difficult to achieve a balance between cleaning, antibacterial, and soothing effects; (2) The dosage form design is unreasonable, and traditional aqueous solutions or creams are prone to flow into the eyes and cause irritation; (3) There is a lack of targeted regulation of the secretion formation mechanism, such as inhibiting excessive mucus secretion or lipid oxidation. Therefore, there is an urgent need to develop a natural plant polyphenol gel specifically for pets, which can optimize the efficacy by combining synergistic ingredients and rely on a suitable gel carrier to achieve a safe and long-lasting secretion inhibition effect while avoiding irritation to the fragile skin around the eyes. Summary of the Invention

[0005] (1) Technical problems solved

[0006] In view of the shortcomings of the existing technology, the present invention provides an eye periocular secretion inhibiting gel containing natural plant polyphenols and a preparation method thereof.

[0007] (2) Technical solution

[0008] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0009] The invention provides a gel for inhibiting periocular secretions containing natural plant polyphenols. The gel is composed of the following components in percentage by mass: 0.5%-2% of epigallocatechin gallate (EGCG), 1%-3% of zinc hyaluronate, 0.01%-0.05% of antimicrobial peptide LL-37, 15%-25% of a gel matrix, and the remainder being deionized water.

[0010] Preferably, the gel is composed of the following components in percentage by mass: EGCG 1.5%, zinc hyaluronate 2.2%, antimicrobial peptide LL-37 0.03%, gel matrix 20%, and the rest is deionized water.

[0011] Furthermore, the gel also includes 0.1%-0.3% of nano-cerium oxide with a particle size of 510 nm.

[0012] Preferably, the gel further comprises 0.21% nano-cerium oxide with a particle size of 510 nm.

[0013] The present invention provides a method for preparing a gel for inhibiting periocular secretions containing natural plant polyphenols, comprising the following steps:

[0014] (1) Disperse Carbomer 940 powder evenly in 80°C constant temperature deionized water and stir until completely swollen;

[0015] (2) After the system is cooled to 40° C., EGCG, zinc hyaluronate, antimicrobial peptide LL-37, and nano-cerium oxide are added in order according to the above mass percentages;

[0016] (3) Adjust the pH to 5.5-6.5 using triethanolamine, then add poloxamer 407 and stir until transparent;

[0017] (4) Filter through a 0.22 μm microporous filter membrane and fill into a light-proof sterile container.

[0018] The present invention provides application of the gel in preparing a product for inhibiting periocular secretions for pets.

[0019] (3) Beneficial effects

[0020] The present invention provides a gel containing natural plant polyphenols to inhibit periocular secretions and its preparation method. This gel innovatively uses epigallocatechin gallate (EGCG) to specifically chelate iron ions, blocking the porphyrin oxidation pathway and reducing pigment deposition. Zinc ions also inhibit the activity of key bacterial metabolic enzymes, inhibiting the growth of pathogens. Furthermore, the antimicrobial peptide LL-37 is used to target and destroy bacterial biofilm structures, achieving a multi-layered tear stain removal effect. Furthermore, a very small amount of nano-cerium oxide is added to enhance the gel's protection against ultraviolet radiation. In vitro tests confirmed a significant synergistic effect between the functional components of the gel, effectively inhibiting the proliferation of Staphylococcus aureus and increasing the porphyrin deposition inhibition rate by 82.10%. Animal test data showed that after two weeks of treatment, the tear stain area around the eyes of the Bichon Frise group was reduced by 75.97%, the McDonald-Shadduck irritation score dropped to 1.87 points, and the corneal fluorescein staining test results of all tested animals were negative, confirming that the gel of the present invention has both excellent tear stain removal effect and good periocular safety, providing a gentle and effective new idea for inhibiting periocular secretions in pets. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Comparison of McDonald-Shadduck eye irritation scores in each group of Bichon Frise dogs; Note: * P<0.05, ** P<0.01.

[0022] Figure 2 Comparison of tear stain area around eyes of Bichon Frise dogs in each group; Note: * P<0.05, ** P<0.01. DETAILED DESCRIPTION

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0024] Example 1

[0025] A gel for suppressing periocular secretions containing natural plant polyphenols is prepared from the following components by weight: 1.5% EGCG, 2.2% zinc hyaluronate, 0.03% antimicrobial peptide LL-37, 0.21% nano-cerium oxide, 20% gel matrix, and the remainder deionized water. The preparation method comprises the following steps:

[0026] (1) Disperse Carbomer 940 powder evenly in 80°C constant temperature deionized water and stir until completely swollen;

[0027] (2) After the system is cooled to 40° C., EGCG, zinc hyaluronate, antimicrobial peptide LL-37, and nano-cerium oxide are added in order according to the above mass percentages;

[0028] (3) Adjust the pH to 5.5-6.5 using triethanolamine, then add poloxamer 407 and stir until transparent;

[0029] (4) Filter through a 0.22 μm microporous filter membrane and fill into a light-proof sterile container.

[0030] Example 2

[0031] The difference between this embodiment and embodiment 1 is that the gel is made of the following components in percentage by mass: EGCG 0.5%, zinc hyaluronate 1%, antimicrobial peptide LL-37 0.01%, nano-cerium oxide 0.1%, gel matrix 15%, and the rest is deionized water.

[0032] Example 3

[0033] The difference between this embodiment and embodiment 1 is that the gel is made of the following components in percentage by mass: EGCG 2%, zinc hyaluronate 3%, antimicrobial peptide LL-37 0.05%, nano-cerium oxide 0.3%, gel matrix 25%, and the remainder is deionized water.

[0034] Example 4

[0035] The difference between this embodiment and embodiment 1 is that the gel is made of the following components in percentage by mass: EGCG 0.7%, zinc hyaluronate 1.2%, antimicrobial peptide LL-37 0.02%, nano-cerium oxide 0.15%, gel matrix 17%, and the rest is deionized water.

[0036] Example 5

[0037] The difference between this embodiment and embodiment 1 is that the gel is made of the following components in percentage by mass: EGCG 1.2%, zinc hyaluronate 2%, antimicrobial peptide LL-37 0.03%, nano-cerium oxide 0.21%, gel matrix 19%, and the rest is deionized water.

[0038] Example 6

[0039] The difference between this embodiment and embodiment 1 is that the gel is made of the following components in percentage by mass: EGCG 1.8%, zinc hyaluronate 2.6%, antimicrobial peptide LL-37 0.04%, nano-cerium oxide 0.27%, gel matrix 23%, and the rest is deionized water.

[0040] Comparative Example 1

[0041] The difference between this comparative example and Example 1 is that the gel does not contain EGCG, and its content is supplemented by deionized water, while the other components and their contents remain unchanged.

[0042] Comparative Example 2

[0043] The difference between this comparative example and Example 1 is that the gel does not contain zinc hyaluronate, and its content is supplemented by deionized water, and the other components and their contents remain unchanged.

[0044] Comparative Example 3

[0045] The difference between this comparative example and Example 1 is that the gel does not contain the antimicrobial peptide LL-37, and its content is supplemented by deionized water, while the other components and their contents remain unchanged.

[0046] Test Example 1

[0047] Antibacterial and anti-porphyrin deposition experiments

[0048] 1 Materials and Methods

[0049] 1.1 Trial Drugs

[0050] The drugs used in Experimental Groups 1-4 corresponded to the gels prepared in Example 1 and Comparative Examples 1-3, respectively. The gels were diluted 10 times and stored in a refrigerator at 4°C for later use.

[0051] 1.2 Test strains

[0052] Staphylococcus aureus (ATCC6538) was purchased from Haibo Biotechnology Co., Ltd. in Qingdao High-Tech Industrial Park.

[0053] 1.3 Antibacterial activity test

[0054] The experiment set up a blank control group and test groups 1-4. Take 1 mL of each of the above test groups 1-4 gels and divide them into test tubes. Use filter paper with uniform texture and punch it into discs with a diameter of 6 mm with a puncher. After sterilization, dry it and soak it in each test tube for 24 hours. The blank control group is soaked in sterile distilled water for later use. In the clean bench, pick Staphylococcus aureus and streak it on LB nutrient agar medium. After incubation at 37°C, pick a single colony and add it to a test tube containing 5 mL of LB broth medium. Incubate it in a constant temperature incubator at 37°C overnight to obtain a bacterial suspension. In the clean bench, dilute the bacterial suspension with LB broth medium and take 100 μL of a concentration of 1×10 8Use a spreading rod to evenly spread a bacterial suspension containing 100 CFU / mL onto the surface of LB nutrient agar. Use flame-sterilized tweezers to pick up the prepared drug-susceptibility paper and place it on the surface of the plate. Incubate upright in a 37°C incubator. After 24 hours, observe and measure the diameter of the inhibition zone. Set up three replicates for each group.

[0055] 1.4 Anti-porphyrin deposition test

[0056] 1.4.1 Artificial tears

[0057] Accurately weigh 0.67g NaCl, 0.02g NaHCO₃, and 0.014g KCl into a beaker. Add 80mL of deionized water and stir on a magnetic stirrer until completely dissolved. Adjust the pH to 7.2-7.4 with 0.1mol / L HCl solution, then slowly add 0.01g bovine serum albumin (BSA). Transfer the mixture to a 100mL volumetric flask and bring to volume with deionized water. Filter the solution through a 0.22μm sterile microporous membrane, aliquot into 5mL sterile centrifuge tubes, and store in a refrigerator at 4°C protected from light until needed.

[0058] 1.4.2 Synthesis of porphyrin

[0059] Accurately weigh 2 mg of protoporphyrin IX powder and dissolve it in 1 mL of phosphate buffer (pH = 7.4). Transfer the prepared solution to a brown glass bottle and store it in a refrigerator at 4°C away from light until use.

[0060] 1.4.3 Porphyrin deposition inhibition rate

[0061] The experiment set up a blank control group, a positive control group, and test groups 1-4, with 3 replicates in each group. Artificial tears and synthetic porphyrin solution were mixed in a volume ratio of 2:1, thoroughly mixed by vortex oscillator, and incubated at 37°C in the dark for 2 hours to form porphyrin-tear complex deposits. A polyester fiber membrane was laid in a 96-well plate, and 100 μL of deposit was added to each well. The blank control group added an equal volume of artificial tears, and then 50 μL of treatment solution was added to each well. Distilled water was added to the blank control group, and 0.1% hydrogen peroxide solution was added to the positive control group. Test groups 1-4 were added to the corresponding test samples. The reaction system was gently shaken and mixed, and then the reaction was kept at 37°C for 30 minutes. Finally, the absorbance was measured at a wavelength of 405 nm using a fluorescence spectrophotometer, and the porphyrin deposition inhibition rate was calculated by the following formula:

[0062] Porphyrin deposition inhibition rate (%) = (A 空白对照组 -A 样品组 ) / A 空白对照组 ×100%

[0063] 1.5 Statistical methods

[0064] GraphPad Prism 8.0 software was used to process the data, and the measured data were expressed as mean ± standard deviation. When all data series showed a normal distribution, ANOVA was used for analysis of variance. When variances were equal, LSD tests were used for comparisons between groups; when variances were unequal, Dunnett's tests were used for comparisons between groups. When one or more data series showed a non-normal distribution, nonparametric tests were used. Differences were considered statistically significant when P < 0.05.

[0065] 2 Results

[0066] 2.1 Antimicrobial activity

[0067] As shown in Table 1, compared with the blank control group, the diameters of the inhibition zones in test groups 1-4 were significantly increased (P<0.01), indicating that the gel of the present invention has significant antibacterial activity against Staphylococcus aureus.

[0068] Table 1 Comparison of inhibition zone diameters in each group

[0069] Grouping Diameter of inhibition zone (mm) Blank control group <![CDATA[6.00±0.00 c ]]> Experimental group 1 <![CDATA[18.30±0.78 a ]]> Experimental Group 2 <![CDATA[13.62±1.67 b <!-- 4 -->]]> Experiment 3 groups <![CDATA[14.64±0.93 b ]]> Experiment 4 groups <![CDATA[12.52±1.08 b ]]>

[0070] Note: Data in the same column with different letters indicate significant differences (P<0.05), and data with the same letters indicate no significant differences (P>0.05).

[0071] 2.2 Porphyrin deposition inhibition rate

[0072] As shown in Table 2, all test groups can significantly inhibit porphyrin deposition, among which the inhibitory effect of test group 1 is the most outstanding, and its inhibition rate is not statistically different from that of the positive control hydrogen peroxide group (P>0.05), indicating that the gel of the present invention can effectively inhibit porphyrin deposition, thereby significantly alleviating the formation of tear stains in pets.

[0073] Table 2 Comparison of porphyrin deposition inhibition rate in each group

[0074]

[0075]

[0076] Note: Data in the same column with different letters indicate significant differences (P<0.05), and data with the same letters indicate no significant differences (P>0.05).

[0077] 3 Conclusion

[0078] The gel of the present invention exhibits significant antibacterial activity against Staphylococcus aureus, effectively inhibiting its proliferation. Furthermore, the gel significantly inhibits porphyrin deposition, effectively alleviating tear stain formation. Group 1 exhibited the best synergistic effect, while groups 2-4 showed a declining efficacy due to the absence of specific components. These experimental results demonstrate that the synergistic effect of the functional components in the gel of the present invention contributes to the tear stain removal effect.

[0079] Test Example 2

[0080] Animal clinical trials

[0081] 1 Materials and Methods

[0082] 1.1 Experimental dogs

[0083] Twenty-four Bichon Frise dogs with severe tear stains were selected, half male and half female, aged 1-4 years old, and weighing 3-6 kg.

[0084] 1.2 Trial Drugs

[0085] The gel of the present invention was prepared according to the scheme described in Example 1.

[0086] 1.3 Treatment

[0087] Twenty-four Bichon Frise dogs were randomly divided into a control group and a treatment group, with 12 dogs in each group. The treatment group used the gel of the present invention daily. The application method was as follows: preliminarily clean the hair around the eyes, apply an appropriate amount of gel to the eyes, massage for 5 minutes, and finally clean the eyes. The application was performed once a day, taking care not to accidentally apply the gel to the eyes. The control group was wiped with normal saline daily for 2 consecutive weeks.

[0088] 1.4 Observation indicators

[0089] 1.4.1 McDonald-Shadduck ocular irritation score

[0090] After the medication, the conjunctival congestion, corneal edema, conjunctival secretions, and corneal opacity of the two groups of Bichon Frise dogs were observed under a slit lamp and scored. The total score was 12 points. The higher the score, the more severe the symptoms.

[0091] 1.4.2 Quantitative assessment of tear stain area

[0092] After the medication, the tear stains of the two groups of Bichon Frise dogs were photographed using a high-definition camera (with fixed light source and angle), and the tear stain area was analyzed using the image analysis software ImageJ.

[0093] 1.5 Statistical methods

[0094] GraphPad Prism 8.0 software was used to process the data, and the measured data were expressed as mean ± standard deviation. When all data series showed a normal distribution, ANOVA was used for analysis of variance. Inter-group comparisons were performed using the LSD test when variances were equal; Dunnett's test was used when variances were unequal. Nonparametric tests were used when one or more data series were non-normally distributed. Inter-group comparisons were performed using the independent sample t-test. Differences were considered statistically significant when P < 0.05.

[0095] 2 Results

[0096] 2.1 McDonald-Shadduck ocular irritation score

[0097] Depend on Figure 1 It can be seen that compared with the control group, the McDonald-Shadduck irritation score of the eyes of the Bichon Frise dogs in the treatment group was significantly reduced (P<0.01), and the corneal fluorescein staining test results were all negative, indicating that the gel of the present invention not only has good eye tolerance, but also can effectively relieve the periocular irritation reaction caused by tear stain accumulation.

[0098] 2.2 Tear stain area

[0099] Depend on Figure 2 It can be seen that compared with the control group, the tear stain area around the eyes of the Bichon Frise dogs in the treatment group was significantly reduced (P<0.01), indicating that the gel of the present invention has a significant effect in removing tear stains, can effectively clear secretions around the eyes, and improve the problem of tear stain deposition.

[0100] 3 Conclusion

[0101] The experimental data above demonstrate that the gel of the present invention demonstrates excellent efficacy in removing periocular secretions and ocular safety in pets. After two weeks of treatment, the tear stain area around the eyes of the Bichon Frise dogs treated with the gel was reduced by 75.97%, and the McDonald-Shadduck irritation score dropped to 1.87. Fluorescein staining of the corneas of all tested animals was negative, demonstrating that the gel is non-irritating to ocular tissue. This invention provides a new, gentle and effective approach for inhibiting periocular secretions in pets.

[0102] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A gel for inhibiting periocular secretions containing natural plant polyphenols, characterized in that: The gel is composed of the following components in percentage by mass: 0.5%-2% of epigallocatechin gallate, 1%-3% of zinc hyaluronate, 0.01%-0.05% of antimicrobial peptide LL-37, 15%-25% of gel matrix, and the remainder being deionized water.

2. The gel for inhibiting periocular secretions containing natural plant polyphenols according to claim 1, characterized in that: The gel is composed of the following components in percentage by mass: 1.5% of epigallocatechin gallate, 2.2% of zinc hyaluronate, 0.03% of antimicrobial peptide LL-37, 20% of gel matrix, and the remainder being deionized water.

3. The gel for inhibiting periocular secretions containing natural plant polyphenols according to claim 1 or 2, characterized in that: The invention also comprises 0.1%-0.3% of nano-cerium oxide with a particle size of 510 nm.

4. The gel for inhibiting periocular secretions containing natural plant polyphenols according to claim 1 or 2, characterized in that: It also includes 0.21% nano-cerium oxide with a particle size of 510 nm.

5. The method for preparing a gel for inhibiting periocular secretions containing natural plant polyphenols according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) Disperse Carbomer 940 powder evenly in 80°C constant temperature deionized water and stir until completely swollen; (2) After the system is cooled to 40° C., epigallocatechin gallate, zinc hyaluronate, antimicrobial peptide LL-37, and nano-cerium oxide are added in order according to the above mass percentages; (3) Adjust the pH to 5.5-6.5 with triethanolamine, then add poloxamer 407 and stir until transparent; (4) Filter through a 0.22 μm microporous filter membrane and fill into a light-proof sterile container.

6. Use of the gel according to any one of claims 1 to 4 in the preparation of a product for inhibiting periocular secretions for pets.

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