Pet tear stain eye drops containing haematococcus pluvialis extract

Abstract

The invention provides pet tear stain eye drops containing haematococcus pluvialis extract, the pH value of the eye drops is 6.8-7.2, the osmotic pressure is 280-320 mOsm / kg, the viscosity is 3-8 mPa.s (25 DEG C), and the eye drops comprise the following components in percentage by mass: 0.05-0.5% of haematococcus pluvialis astaxanthin extract (based on the astaxanthin content being greater than or equal to 5%), 0.05-0.5% of a pH regulator (based on the astaxanthin content being greater than or equal to 5%), and the balance of water. 1%-3% of an anti-inflammatory plant compound; 0.5%-1.2% of an isoosmotic adjusting agent; 0.2%-0.8% of a nano carrier stabilizer; 0.01%-0.1% of a pH buffer agent; compared with the prior art, the invention has the following beneficial effects: the corneal permeability of the astaxanthin is increased to 82% (measured by a Franz diffusion cell) through PLGA coating, and is increased by 5.5 times compared with the corneal permeability of the astaxanthin in a free state (15%); natural tocopherol is reserved through supercritical CO2 extraction (the content of the natural tocopherol is larger than or equal to 0.5% through HPLC detection), and the retention rate of the astaxanthin stored at 40 DEG C for 90 days is larger than or equal to 90% through Calendula quercitrin inhibits the expression of IL-6 (enzyme-linked immunosorbent assay (ELISA) detection is reduced by 76%), and chamomile bisabolol blocks a COX-2 pathway.

Description

Technical Field

[0001] The invention belongs to the technical field of pet eye care, and in particular relates to pet tear mark eye drops containing Haematococcus pluvialis extract. Background Art

[0002] As people become more accepting of pets, the market size of pet products continues to expand. For example, pet tear stain eye drops are commonly used by cats and dogs. However, current pet tear stain eye drops have the following technical defects: chemical bleaching ingredients (such as hydrogen peroxide) cause damage to corneal epithelial cells due to their strong oxidizing properties, and the excessive release of free radicals in their mechanism of action causes conjunctival congestion rates ≥40%. Although antibiotic products (such as chloramphenicol) can inhibit bacteria in the short term, long-term use leads to a 55% increase in the detection rate of drug-resistant strains. In addition, the toxicity of antibiotics to limbal stem cells reduces the ocular surface repair capacity by 37%.

[0003] Natural plant extracts (such as calendula) have a corneal permeability of ≤15% (as measured by Franz diffusion cell) due to their large molecular weight (>50 kDa) and poor lipid solubility. This is due to the failure to address the transmembrane transport barrier. While existing technologies using propylene glycol as a permeation enhancer can increase permeability to 30%, its toxicity to goblet cells reduces tear secretion by 28%.

[0004] Due to chemical instability, antioxidants such as astaxanthin lose ≥70% of their activity after 30 days of storage at room temperature. This is attributed to photooxidation and molecular isomerization in the liquid environment (HPLC analysis shows that the proportion of trans-astaxanthin has dropped from 95% to 42%). Although existing microencapsulation technology has increased the retention rate to 60%, the capsule wall material (such as gelatin) reacts with tear electrolytes to produce floccules, increasing the risk of tear duct blockage by 25%.

[0005] Isotonicity regulation relies on a traditional borate buffer system. While this maintains an osmotic pressure of 290 mOsm / kg, borate binds to corneal mucin, causing a burning sensation (Draize score ≥ 2.5). Using sodium chloride alone for regulation disrupts the tear film's ion balance due to fluctuations in sodium ion concentration (>20 mmol / L). The combined effects of these deficiencies make existing products unable to meet clinical needs. Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a pet tear stain eye drops containing Haematococcus pluvialis extract to solve the problems raised in the above background technology.

[0007] The present invention is achieved through the following technical solution: a pet tear stain eye drop containing Haematococcus pluvialis extract, wherein the eye drop has a pH value of 6.8-7.2, an osmotic pressure of 280-320 mOsm / kg, and a viscosity of 3-8 mPa·s (25°C), and comprises the following components in percentage by weight:

[0008] Haematococcus pluvialis astaxanthin extract 0.05% to 0.5% (calculated based on astaxanthin content ≥ 5%);

[0009] Anti-inflammatory plant complex 1% to 3%;

[0010] Isotonicity regulator 0.5% to 1.2%;

[0011] Nanocarrier stabilizer 0.2% to 0.8%;

[0012] pH buffer 0.01% to 0.1%;

[0013] The balance is ultrapure water (conductivity ≤ 5 μS / cm).

[0014] As a preferred embodiment, the astaxanthin extract of Haematococcus pluvialis is prepared by a supercritical CO2 extraction process, the conditions being: extraction pressure 35-45 MPa, temperature 40-50°C, entrainer ethanol-ethyl acetate (volume ratio 3:1), extraction time 120-180 min, and astaxanthin extraction rate ≥92%.

[0015] As a preferred embodiment, the oxidative stability index (OSI) of the Haematococcus pluvialis astaxanthin extract is ≥20h, and the proportion of trans-astaxanthin is ≥95%.

[0016] As a preferred embodiment, the anti-inflammatory plant compound is composed of calendula extract, chamomile extract and aloe polysaccharide in a weight ratio of (2-4): (1-3): (0.5-1.5), wherein:

[0017] The quercetin content in the Calendula officinalis extract is ≥8%;

[0018] Bisabolol ≥1.2% in chamomile extract;

[0019] The molecular weight of aloe polysaccharide is 10-50 kDa (determined by GPC).

[0020] As a preferred embodiment, the nanocarrier stabilizer is astaxanthin nanoparticles coated with polylactic acid-glycolic acid copolymer, with a particle size D90 ≤ 200 nm and an encapsulation efficiency ≥ 85%.

[0021] As a preferred embodiment, the isotonicity regulator is a complex of trehalose and sodium chloride, with a mass ratio of (3-5):1, and the sodium ion concentration in the solution is ≤15 mmol / L.

[0022] As a preferred embodiment, the pH buffer is a Tris-HCl buffer system with a buffer capacity of β=0.01 to 0.03 mol / L / pH, and is sterilized by a 0.22 μm filter membrane after preparation.

[0023] As a preferred embodiment, the method is prepared by the following steps:

[0024] a) dissolving the astaxanthin extract of Haematococcus pluvialis and PLGA in acetone and homogenizing under high pressure (800-1200 bar) to form a nanosuspension;

[0025] b) dissolving the anti-inflammatory plant complex and trehalose in 60° C. ultrapure water, cooling to 30° C., and then adding sodium chloride;

[0026] c) mixing the nanosuspension with water and magnetically stirring (300-500 rpm) to remove residual acetone;

[0027] d) Adjust the pH to 7.0±0.1, filter through 0.22 μm sterile filtration, and then fill with nitrogen.

[0028] In step a), the high pressure homogenization is repeated 5 to 8 times, and the mixture is cooled to 10 to 15° C. between each time. The intrinsic viscosity of PLGA is 0.2 to 0.4 dL / g.

[0029] After adopting the above technical scheme, the beneficial effects of the present invention are as follows: PLGA coating increases the corneal permeability of astaxanthin to 82% (determined by Franz diffusion cell), which is 5.5 times higher than the free state (15%); supercritical CO2 extraction retains natural tocopherol (HPLC detection ≥0.5%), and synergistically with trehalose, the retention rate of astaxanthin stored at 40°C for 90 days is ≥90%; marigold quercetin inhibits IL-6 expression (ELISA detection decreases by 76%), and chamomile bisabolol blocks the COX-2 pathway (Western blot verification); trehalose-sodium chloride composite regulation makes the solution osmotic pressure precisely match tears (295mOsm / kg), avoiding the irritation of traditional borate buffer. DETAILED DESCRIPTION

[0030] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0031] The present invention provides a technical solution: a pet tear stain eye drop containing a Haematococcus pluvialis extract, wherein the eye drop has a pH value of 6.8 to 7.2, an osmotic pressure of 280 to 320 mOsm / kg, and a viscosity of 3 to 8 mPa·s (25° C.), and comprises the following components in percentage by mass:

[0032] Haematococcus pluvialis astaxanthin extract 0.05% to 0.5% (calculated based on astaxanthin content ≥ 5%);

[0033] Anti-inflammatory plant complex 1% to 3%;

[0034] Isotonicity regulator 0.5% to 1.2%;

[0035] Nanocarrier stabilizer 0.2% to 0.8%;

[0036] pH buffer 0.01% to 0.1%;

[0037] The balance is ultrapure water (conductivity ≤ 5 μS / cm).

[0038] The astaxanthin extract of Haematococcus pluvialis is prepared by a supercritical CO2 extraction process under the following conditions: extraction pressure of 35 to 45 MPa, temperature of 40 to 50°C, entrainer of ethanol-ethyl acetate (volume ratio of 3:1), extraction time of 120 to 180 minutes, and astaxanthin extraction rate of ≥92%.

[0039] The oxidative stability index (OSI) of the Haematococcus pluvialis astaxanthin extract is ≥20h, and the proportion of trans-astaxanthin is ≥95%.

[0040] The anti-inflammatory plant complex is composed of calendula extract, chamomile extract and aloe polysaccharide in a weight ratio of (2-4): (1-3): (0.5-1.5), wherein:

[0041] The quercetin content in the Calendula officinalis extract is ≥8%;

[0042] Bisabolol ≥1.2% in chamomile extract;

[0043] The molecular weight of aloe polysaccharide is 10-50 kDa (determined by GPC).

[0044] The nano-carrier stabilizer is astaxanthin nanoparticles coated with polylactic acid-glycolic acid copolymer, with a particle size D90 of ≤200 nm and an encapsulation rate of ≥85%.

[0045] The isotonicity regulator is a complex of trehalose and sodium chloride, with a mass ratio of (3-5):1, and a sodium ion concentration in the solution is ≤15 mmol / L.

[0046] The pH buffer is a Tris-HCl buffer system with a buffer capacity of β=0.01-0.03 mol / L / pH, and is sterilized by a 0.22 μm filter membrane after preparation.

[0047] Prepared by the following steps:

[0048] a) dissolving the astaxanthin extract of Haematococcus pluvialis and PLGA in acetone and homogenizing under high pressure (800-1200 bar) to form a nanosuspension;

[0049] b) dissolving the anti-inflammatory plant complex and trehalose in 60° C. ultrapure water, cooling to 30° C., and then adding sodium chloride;

[0050] c) mixing the nanosuspension with water and magnetically stirring (300-500 rpm) to remove residual acetone;

[0051] d) Adjust the pH to 7.0±0.1, filter through 0.22 μm sterile filtration, and then fill with nitrogen.

[0052] In step a), the high pressure homogenization is repeated 5 to 8 times, and the mixture is cooled to 10 to 15° C. between each time. The intrinsic viscosity of PLGA is 0.2 to 0.4 dL / g.

[0053] As an embodiment of the present invention: Example 1,

[0054] The eye drops have a pH value of 6.8 to 7.2, an osmotic pressure of 280 to 320 mOsm / kg, and a viscosity of 3 to 8 mPa·s (25° C.), and contain the following components in percentage by weight:

[0055] Haematococcus pluvialis astaxanthin extract 0.2% (astaxanthin content 5.8%)

[0056] Anti-inflammatory complex 2.5% (Calendula:Chamomile:Aloe Vera=3:2:1)

[0057] Trehalose 0.9% + Sodium Chloride 0.3%

[0058] PLGA nanoparticles 0.5% (particle size D90 = 185 nm)

[0059] Tris buffer 0.05%

[0060] Ultrapure water (conductivity 4 μS / cm) balance.

[0061] Prepared by the following steps:

[0062] PLGA nanoparticles (intrinsic viscosity 0.32 dL / g) and astaxanthin extract from Haematococcus pluvialis were dissolved in acetone at a ratio of 10:1 and homogenized six times at 1200 bar (with intermittent cooling to 12°C). The acetone was removed by rotary evaporation until the residual amount was ≤30 ppm. Calendula, chamomile, and aloe vera extracts and trehalose were dissolved at 60°C. After cooling, sodium chloride was added, and the nanosuspension was mixed with the aqueous phase and magnetically stirred (400 rpm × 30 min). The mixture was filtered through a 0.22 μm PVDF membrane and filled with nitrogen.

[0063] The tear mark eye drops of Example 1 were tested and compared with the comparative example (commercially available tear mark eye drops). The comparison results are shown in Table 1.

[0064]

[0065] Table 1

[0066] Through Example 1, it can be seen that the advantages of the pet tear mark eye drops of the present invention are:

[0067] 1. Supercritical CO2 extraction: extraction pressure of 40MPa, entrainer ethanol-ethyl acetate (3:1) makes the astaxanthin extraction rate reach 92% (compared with 65% of traditional organic solvent extraction method), and retains natural tocopherol (HPLC detection 0.58%), and synergistic antioxidant effect makes the OSI value reach 23h (the background technology microencapsulation method is only 12h);

[0068] PLGA nanocoating: Nanoparticles with a particle size of 185 nm increase corneal permeability to 8.25 μg / cm 2 / h (the free astaxanthin in the background art is only 1.32 μg), and the hydrophobic core of PLGA prevents astaxanthin from isomerizing (the trans configuration is retained ≥95%, while the background art drops to 42% after liquid storage).

[0069] 2. Calendula-Chamomile-Aloe Vera Triple Complex:

[0070] Quercetin inhibits IL-6 expression (a 76% decrease in ELISA, compared to a 45% decrease in single calendula extract); bisabolol blocks the COX-2 pathway (an 82% decrease in Western blot grayscale value, compared to a 90% decrease in ibuprofen-like drugs but causing corneal damage); 10-50kDa aloe polysaccharide promotes corneal repair (scratch tests showed a 2.1-fold increase in healing speed).

[0071] 3. Trehalose-sodium chloride isotonic system: sodium ion concentration is controlled at 12 mmol / L (ICP-MS), avoiding the burning sensation of traditional borates (Draize score 0.5 vs 2.8);

[0072] The nanocarrier avoids the toxicity of penetration enhancers: without using chemical penetration enhancers such as propylene glycol, the goblet cell survival rate is ≥95% (Calcein-AM staining method, background technology penetration enhancer group ≤72%).

[0073] Example 2,

[0074] Adjust the raw materials and methods, specifically:

[0075] During homogenization, the homogenization pressure was reduced to 600 bar; and PLGA coating was not used.

[0076] The tear mark eye drops of Example 1 were compared with the tear mark eye drops of Example 2. The results are shown in Table 2.

[0077] index Example 1 Example 2 Cause Analysis Nanoparticle size D90 185nm 650nm Insufficient homogenization pressure causes aggregation Corneal permeability 8.25 1.98 Too large particle size hinders penetration Storage stability (precipitation rate) 0％ 5.7％ Uncoated leads to oxidation precipitation

[0078] Table 2

[0079] Through Example 2, it can be seen that the necessity of high-pressure homogenization process

[0080] At a homogenization pressure of 600 bar, the nanoparticle size D90 = 650 nm (compared to 185 nm in Example 1), resulting in a corneal permeability drop to 1.98 μg / cm 2 / h, verifying the technical critical point of the pressure range of 35-45MPa in claim 2; the precipitation rate of uncoated astaxanthin reached 5.7% (laser scattering method), proving the decisive role of PLGA encapsulation on physical stability (the background technology gelatin encapsulation precipitation rate is 3.2%, but it causes tear duct blockage).

[0081] After omitting PLGA, the lack of nanocarriers results in the inability to target the release of anti-inflammatory ingredients:

[0082] The IL-6 inhibition rate dropped to 34% (76% in Example 1);

[0083] The tear stain reduction rate decreased from 78.6% to 52.1%, demonstrating the carrier-ingredient synergistic mechanism.

[0084] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A pet tear stain eye drops containing Haematococcus pluvialis extract, characterized in that: The eye drops have a pH value of 6.8 to 7.2, an osmotic pressure of 280 to 320 mOsm / kg, and a viscosity of 3 to 8 mPa·s (25° C.), and contain the following components in percentage by weight: Haematococcus pluvialis astaxanthin extract 0.05% to 0.5% (calculated based on astaxanthin content ≥ 5%); Anti-inflammatory plant complex 1% to 3%; Isotonicity regulator 0.5% to 1.2%; Nanocarrier stabilizer 0.2% to 0.8%; pH buffer 0.01% to 0.1%; The balance is ultrapure water (conductivity ≤ 5 μS / cm).

2. The pet tear stain eye drops containing the Haematococcus pluvialis extract according to claim 1, characterized in that: The astaxanthin extract of Haematococcus pluvialis is prepared by a supercritical CO2 extraction process under the following conditions: extraction pressure of 35 to 45 MPa, temperature of 40 to 50°C, entrainer of ethanol-ethyl acetate (volume ratio of 3:1), extraction time of 120 to 180 minutes, and astaxanthin extraction rate of ≥92%.

3. The pet tear stain eye drops containing the Haematococcus pluvialis extract according to claim 2, characterized in that: The oxidative stability index (OSI) of the Haematococcus pluvialis astaxanthin extract is ≥20h, and the proportion of trans-astaxanthin is ≥95%.

4. The pet tear stain eye drops containing the Haematococcus pluvialis extract according to claim 1, characterized in that: The anti-inflammatory plant complex is composed of calendula extract, chamomile extract and aloe polysaccharide in a weight ratio of (2-4): (1-3): (0.5-1.5), wherein: The quercetin content in the Calendula officinalis extract is ≥8%; Bisabolol ≥1.2% in chamomile extract; The molecular weight of aloe polysaccharide is 10-50 kDa (determined by GPC).

5. The pet tear stain eye drops containing the Haematococcus pluvialis extract according to claim 1, characterized in that: The nano-carrier stabilizer is astaxanthin nanoparticles coated with polylactic acid-glycolic acid copolymer, with a particle size D90 of ≤200 nm and an encapsulation rate of ≥85%.

6. The pet tear stain eye drops containing the Haematococcus pluvialis extract according to claim 1, characterized in that: The isotonicity regulator is a complex of trehalose and sodium chloride, with a mass ratio of (3-5):1, and a sodium ion concentration in the solution is ≤15 mmol / L.

7. The pet tear stain eye drops containing the Haematococcus pluvialis extract according to claim 1, characterized in that: The pH buffer is a Tris-HCl buffer system with a buffer capacity of β=0.01-0.03 mol / L / pH, and is sterilized by a 0.22 μm filter membrane after preparation.

8. The pet tear stain eye drops containing the Haematococcus pluvialis extract according to claim 1, characterized in that: Prepared by the following steps: a) dissolving the astaxanthin extract of Haematococcus pluvialis and PLGA in acetone and homogenizing under high pressure (800-1200 bar) to form a nanosuspension; b) dissolving the anti-inflammatory plant complex and trehalose in 60° C. ultrapure water, cooling to 30° C., and then adding sodium chloride; c) mixing the nanosuspension with water and magnetically stirring (300-500 rpm) to remove residual acetone; d) Adjust the pH to 7.0±0.1, filter through 0.22 μm sterile filtration, and then fill with nitrogen.

9. The pet tear stain eye drops containing the Haematococcus pluvialis extract according to claim 8, characterized in that: In step a), the high pressure homogenization is repeated 5 to 8 times, and the mixture is cooled to 10 to 15° C. between each time. The intrinsic viscosity of PLGA is 0.2 to 0.4 dL / g.

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