Pet anti-aging additive based on compound functional components and preparation method of pet anti-aging additive

Abstract

The invention provides a pet anti-aging additive based on compound functional components and a preparation method thereof, and relates to the technical field of pet foods.The additive takes arabinoxylan, micrococcus oil and nano traditional Chinese medicine extracts as matrixes to construct a multi-dimensional anti-inflammatory barrier system, and the multi-dimensional anti-inflammatory barrier system is prepared into the pet anti-aging additive based on the compound functional components. The proanthocyanidins and the micrococcus oil are adopted to form a neuroprotection synergistic interaction system, and meanwhile, a microencapsulation process is combined, so that the bioavailability is improved, and a multi-dimensional anti-aging mechanism of resisting oxidation, regulating and controlling senescence-related markers and resisting inflammation is realized; in-vitro experiments show that the additive can effectively inhibit the expression of SA-beta-gal in canine fibroblasts, and can reduce the MDA content and relieve oxidative damage by improving the activity of SOD and GSH-Px; in-vivo experiments show that the additive can effectively reduce the CCDR score of a test dog, obviously improve the cognitive function of the test dog, improve the NAD + level of plasma, reduce the levels of TNF-alpha, IL-1beta and IL-6 in joint synovial fluid and exert the anti-aging effect, and a safe and efficient new scheme is provided for delaying pet aging.

Description

Technical Field

[0001] The present invention relates to the technical field of pet food, in particular to a pet anti-aging additive based on composite functional ingredients and a preparation method thereof. Background Art

[0002] In recent years, with the improvement of pet-keeping concepts and the development of veterinary medicine, the average lifespan of pets has been significantly extended, and the aging problem has become increasingly prominent. Studies have shown that companion animals such as dogs and cats are prone to oxidative stress damage, mitochondrial dysfunction, telomere shortening, and chronic inflammatory responses after entering the elderly stage, which can lead to aging-related health problems such as metabolic slowdown, decreased immune function, joint degeneration, skin and hair aging, and cognitive decline. At present, pet anti-aging products mainly rely on single antioxidants such as vitamin C, vitamin E, coenzyme Q10, etc. or natural plant extracts such as resveratrol, curcumin, and grape seed polyphenols. However, the existing technology still has the following limitations: (1) The efficacy of a single ingredient is limited. Traditional antioxidants often only target a certain pathway of the aging mechanism and lack multi-target synergistic regulation, making it difficult to fully delay the aging process; (2) Low bioavailability. Most antioxidant ingredients are easily affected by the pH environment or digestive enzymes in the pet's gastrointestinal tract, resulting in insufficient absorption rate and difficulty in achieving effective blood drug concentrations. (3) Poor palatability. Some anti-aging ingredients (such as curcumin) affect pets’ compliance with feeding due to odor or taste issues. Therefore, there is an urgent need to develop a pet anti-aging additive with multi-component synergy and efficient preparation process to address the shortcomings of existing technologies such as single ingredients, low absorption rate, and insufficient safety. Summary of the Invention

[0003] (1) Technical problems solved

[0004] In view of the deficiencies in the prior art, the present invention provides a pet anti-aging additive based on composite functional ingredients and a preparation method thereof.

[0005] (2) Technical solution

[0006] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0007] The invention provides an anti-aging additive for pets based on composite functional ingredients. The additive is prepared from the following active ingredients in percentage by weight: 20-35 parts of pseudo-nanochloridia oil, 15-30 parts of proanthocyanidins, 10-25 parts of arabinoxylan, 5-20 parts of N-acetylglucosamine, 12 parts of Bifidobacterium longum M-633, 6-25 parts of nano-traditional Chinese medicine extracts, and 1-10 parts of maqui berry anthocyanins.

[0008] Furthermore, the additive is made of the following active ingredients in percentage by weight: 25-30 parts of pseudo-nanochloridia oil, 20-25 parts of proanthocyanidins, 15-20 parts of arabinoxylan, 10-15 parts of N-acetylglucosamine, 8 parts of Bifidobacterium longum M-635, 10-20 parts of nano-traditional Chinese medicine extract, and 2-7 parts of maqui berry anthocyanins.

[0009] Furthermore, the additive is made of the following active ingredients in percentage by weight: 25 parts of pseudo-nanochloridia oil, 20 parts of proanthocyanidins, 15 parts of arabinoxylan, 12 parts of N-acetylglucosamine, 37 parts of Bifidobacterium longum M-63, 15 parts of nano-traditional Chinese medicine extract, and 3 parts of maqui berry anthocyanins.

[0010] Furthermore, the nano-TCM extract is compounded from the following raw materials in a mass ratio: frankincense: yam: mistletoe: locust=3-10:2-8:1-3:1-3.

[0011] Furthermore, the nano-TCM extract is compounded from the following raw materials in a mass ratio: frankincense: yam: mistletoe: locust = 7:5:2:1.

[0012] The present invention provides a method for preparing a pet anti-aging additive based on composite functional ingredients, comprising the following steps:

[0013] (1) Preparation of nano-TCM extracts: Frankincense, Chinese yam, mistletoe and locust were weighed separately, cleaned and pulverized into fine powder; 70% ethanol solution was used as the extraction medium for ultrasonic extraction and then vacuum rotary evaporation was used to obtain the extract; the extract was dispersed in olive oil at a mass ratio of 1:5 to form an oil phase, and 2% Tween-80 was dissolved in water as the aqueous phase, and the nanoemulsion was prepared by high-speed shear emulsification and high-pressure homogenization.

[0014] (2) Composite liposome encapsulation: Soybean lecithin and cholesterol were mixed in a 2:1 molar ratio, dissolved in methanol, and subjected to a rotary evaporator to form a uniform lipid film; the lipid film was re-dissolved in PBS buffer, and proanthocyanidins and maqui berry anthocyanins were added. After ultrasonic hydration at 37°C for 30-60 min, nanoliposomes were obtained by extrusion through a 0.1 μm polycarbonate membrane.

[0015] (3) After homogenizing the above-mentioned nano-TCM extract, nanoliposomes and pseudomicrocystis oil, arabinoxylan and N-acetylglucosamine were added, and microencapsulated by low-temperature spray drying. Subsequently, the additive of the present invention was obtained by mixing with Bifidobacterium longum M-63 freeze-dried powder under sterile conditions.

[0016] Furthermore, in the step (1), the power of the ultrasonic extraction is 40 kHz, the temperature is 40-55° C., the time is 20-30 min, the speed of the high-speed shear emulsification is 10,000 rpm, the time is 5-15 min, and the pressure of the high-pressure homogenizer is 100 MPa; and the process parameters of the low-temperature spray drying in the step (3) are an inlet air temperature of 120° C., an outlet air temperature of 60° C., and an atomization pressure of 0.3 MPa.

[0017] The present invention provides application of the additive in preparing a product for delaying pet aging.

[0018] Furthermore, during use, the additive is added in an amount of 2-5% of the complete grain.

[0019] (3) Beneficial effects

[0020] The present invention provides a pet anti-aging additive based on composite functional ingredients and a preparation method thereof. The additive uses arabinoxylan, pseudomicrocystis oil, and nano-traditional Chinese medicine extracts as a matrix, and constructs a multi-dimensional intestinal-joint-brain anti-inflammatory barrier system through multi-component collaboration; uses proanthocyanidins and pseudomicrocystis oil to form a neuroprotective synergistic enhancement system; and is compounded with Bifidobacterium longum M-63 strain to activate the immune regulation pathway; and at the same time, combined with the microencapsulation process, significantly improves the bioavailability of the active ingredients, and realizes a multi-dimensional anti-aging mechanism of anti-oxidative stress, regulation of aging-related markers, and inhibition of inflammatory factor networks. The results of in vitro experiments show that the additive of the present invention can effectively inhibit the expression of SA-β-gal, a marker of canine fibroblast aging, and reduce oxidative damage by significantly increasing the activity of antioxidant enzymes SOD and GSH-Px and reducing MDA content; in vivo experiments further verify that the additive of the present invention can effectively reduce the CCDR score of the test dogs, significantly improve the cognitive function of the test dogs, and can also effectively increase plasma NAD + levels, and reduces the levels of inflammatory factors TNF-α, IL-1β, and IL-6 in the synovial fluid of the joints, thereby exerting anti-aging effects and providing a safe and efficient new solution for delaying the aging of pets. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Comparison of antioxidant indexes in each group. Note: Compared with the blank control group, # P<0.05, ## P<0.01; compared with the model control group, * P<0.05, ** P<0.01. DETAILED DESCRIPTION

[0022] 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.

[0023] Example 1

[0024] A pet anti-aging additive based on a composite functional ingredient, comprising the following active ingredients in percentage by weight: 25 parts of pseudo-nanochloridia oil, 20 parts of proanthocyanidins, 15 parts of arabinoxylan, 12 parts of N-acetylglucosamine, 7 parts of Bifidobacterium longum M-63, 15 parts of a nano-herbal extract from traditional Chinese medicine, and 3 parts of maqui berry anthocyanins. The nano-herbal extract is compounded from the following raw materials in a mass ratio of 7:5:2:1: frankincense, yam, mistletoe, and locust.

[0025] The preparation method of the additive comprises the following steps:

[0026] (1) Preparation of nano-TCM extracts: Frankincense, Chinese yam, mistletoe and locust were weighed separately, cleaned and pulverized into fine powder; 70% ethanol solution was used as the extraction medium for ultrasonic extraction and then vacuum rotary evaporation was used to obtain the extract; the extract was dispersed in olive oil at a mass ratio of 1:5 to form an oil phase, and 2% Tween-80 was dissolved in water as the aqueous phase, and the nanoemulsion was prepared by high-speed shear emulsification and high-pressure homogenization.

[0027] (2) Composite liposome encapsulation: Soybean lecithin and cholesterol were mixed in a 2:1 molar ratio, dissolved in methanol, and subjected to a rotary evaporator to form a uniform lipid film; the lipid film was re-dissolved in PBS buffer, and proanthocyanidins and maqui berry anthocyanins were added. After ultrasonic hydration at 37°C for 30-60 min, nanoliposomes were obtained by extrusion through a 0.1 μm polycarbonate membrane.

[0028] (3) After homogenizing the above-mentioned nano-TCM extract, nanoliposomes and pseudomicrocystis oil, arabinoxylan and N-acetylglucosamine were added, and microencapsulated by low-temperature spray drying. Subsequently, the additive of the present invention was obtained by mixing with Bifidobacterium longum M-63 freeze-dried powder under sterile conditions.

[0029] In the step (1), the power of the ultrasonic extraction is 40 kHz, the temperature is 40-55° C., the time is 20-30 min, the speed of the high-speed shear emulsification is 10,000 rpm, the time is 5-15 min, and the pressure of the high-pressure homogenizer is 100 MPa; and the process parameters of the low-temperature spray drying in the step (3) are an inlet air temperature of 120° C., an outlet air temperature of 60° C., and an atomization pressure of 0.3 MPa.

[0030] Example 2

[0031] This embodiment differs from embodiment 1 in that the additive is made of the following active ingredients in percentage by weight: 20 parts of pseudo-nanochloridia oil, 15 parts of proanthocyanidins, 10 parts of arabinoxylan, 5 parts of N-acetylglucosamine, 3 parts of Bifidobacterium longum M-63, 6 parts of nano-TCM extract, and 1 part of maqui anthocyanin. The nano-TCM extract is compounded from the following raw materials in a mass ratio of frankincense: Chinese yam: mistletoe: locust tree = 3:2:1:1.

[0032] Example 3

[0033] This embodiment differs from embodiment 1 in that the additive is made of the following active ingredients in percentage by weight: 32 parts of pseudo-nanochloridia oil, 28 parts of proanthocyanidins, 22 parts of arabinoxylan, 18 parts of N-acetylglucosamine, 11 parts of Bifidobacterium longum M-63, 22 parts of nano-TCM extract, and 7 parts of maqui anthocyanins. The nano-TCM extract is compounded from the following raw materials in a mass ratio of 9:8:3:3:1.

[0034] Example 4

[0035] This embodiment differs from embodiment 1 in that the additive is made of the following active ingredients in percentage by weight: 22 parts of pseudo-nanochloridia oil, 18 parts of proanthocyanidins, 12 parts of arabinoxylan, 7 parts of N-acetylglucosamine, 5 parts of Bifidobacterium longum M-63, 8 parts of nano-TCM extract, and 2 parts of maqui berry anthocyanins. The nano-TCM extract is compounded from the following raw materials in a mass ratio of frankincense: Chinese yam: mistletoe: locust tree = 5:7:2:3.

[0036] Example 5

[0037] This embodiment differs from embodiment 1 in that the additive is made of the following active ingredients in percentage by weight: 35 parts of pseudo-nanochloridia oil, 30 parts of proanthocyanidins, 25 parts of arabinoxylan, 20 parts of N-acetylglucosamine, 12 parts of Bifidobacterium longum M-63, 25 parts of nano-TCM extract, and 10 parts of maqui anthocyanins. The nano-TCM extract is compounded from the following raw materials in a mass ratio of frankincense: Chinese yam: mistletoe: locust tree = 10:8:3:3.

[0038] Example 6

[0039] This embodiment differs from embodiment 1 in that the additive is made of the following active ingredients in percentage by weight: 30 parts of pseudo-nanochloridia oil, 25 parts of proanthocyanidins, 20 parts of arabinoxylan, 17 parts of N-acetylglucosamine, 39 parts of Bifidobacterium longum M-6, 20 parts of nano-TCM extract, and 5 parts of maqui anthocyanins. The nano-TCM extract is compounded from the following raw materials in a mass ratio of 8:7:3:3:1.

[0040] Example 7

[0041] This embodiment differs from embodiment 1 in that the additive is made of the following active ingredients in percentage by weight: 28 parts of pseudo-nanochloridia oil, 22 parts of proanthocyanidins, 18 parts of arabinoxylan, 15 parts of N-acetylglucosamine, 38 parts of Bifidobacterium longum M-6, 18 parts of nano-TCM extract, and 4 parts of maqui anthocyanins. The nano-TCM extract is compounded from the following raw materials in a mass ratio of: frankincense: yam: mistletoe: locust = 6:8:3:2.

[0042] Comparative Example 1

[0043] The difference between this comparative example and Example 1 is that its preparation method comprises the following steps:

[0044] (1) Weighing each raw material in a mass ratio of frankincense: Chinese yam: mistletoe: locust = 7:5:2:1, cleaning and crushing into fine powder, using 70% ethanol solution as an extraction medium for ultrasonic extraction, and then vacuum rotary evaporation to obtain a Chinese medicine extract;

[0045] (2) The Chinese herbal medicine extract, proanthocyanidins, and pseudomicrocystis oil are homogeneously mixed, and then arabinoxylan, N-acetylglucosamine, and maqui berry anthocyanin are added, and the mixture is dried using a low-temperature spray drying technique. The mixture is then mixed with freeze-dried powder of Bifidobacterium longum M-63 under sterile conditions to obtain the additive of the present invention.

[0046] Test Example 1

[0047] 1 Materials and Methods

[0048] 1.1 Trial Drugs

[0049] Test Group 1: 25 parts of Nannochloropsis oil, 20 parts of proanthocyanidins, 15 parts of arabinoxylan, 12 parts of N-acetylglucosamine, 7 parts of Bifidobacterium longum M-63, 15 parts of a traditional Chinese medicine extract, and 3 parts of maqui berry anthocyanins. The traditional Chinese medicine extract was prepared by combining the following ingredients in a mass ratio: frankincense, Chinese yam, mistletoe, and locust (7:5:2:1).

[0050] Test Group 2: 15 parts of arabinoxylan, 12 parts of N-acetylglucosamine, 7 parts of Bifidobacterium longum M-63, 15 parts of a traditional Chinese medicine extract, and 3 parts of maqui berry anthocyanins. The traditional Chinese medicine extract was compounded with the following raw materials in a mass ratio of 7:5:2:1: frankincense, yam, mistletoe, and locust.

[0051] Experiment 3: 25 parts of Nannochloropsis oil, 20 parts of proanthocyanidins, 15 parts of arabinoxylan, 12 parts of N-acetylglucosamine, 7 parts of Bifidobacterium longum M-63, and 3 parts of maqui berry anthocyanins.

[0052] Test group 4: 25 parts of Nannochloropsis oil, 20 parts of proanthocyanidins, 15 parts of arabinoxylan, 12 parts of N-acetylglucosamine, 7 parts of Bifidobacterium longum M-63, and 15 parts of a traditional Chinese medicine extract. The traditional Chinese medicine extract was compounded from the following raw materials in a mass ratio of 7:5:2:1: frankincense, yam, locust, and so on.

[0053] Test Group 5: 25 parts of Nannochloropsis oil, 20 parts of proanthocyanidins, 15 parts of arabinoxylan, 12 parts of N-acetylglucosamine, 7 parts of Bifidobacterium longum M-63, 15 parts of a traditional Chinese medicine extract, and 3 parts of maqui berry anthocyanins. The traditional Chinese medicine extract was compounded with the following raw materials in a mass ratio of 7:5: frankincense and yam.

[0054] Preparation method: frankincense, yam, mistletoe, and locust are weighed respectively according to the above weight ratio, cleaned and crushed into fine powder, ultrasonically extracted using 70% ethanol solution as an extraction medium, and then vacuum rotary evaporation is performed to obtain a traditional Chinese medicine extract; the traditional Chinese medicine extract, proanthocyanidins, pseudo-nanochloris oil, and maqui anthocyanins are homogenously mixed according to the above weight percentages, and then arabinoxylan and N-acetylglucosamine are added, and the mixture is dried using a low-temperature spray drying technology. Subsequently, the mixture is mixed with freeze-dried powder of Bifidobacterium longum M-63 under sterile conditions to obtain additives for test groups 1-5.

[0055] 1.2 Primary isolation and culture of canine skin fibroblasts

[0056] Healthy adult beagle dogs (15-18 months old) were selected, and full-thickness skin samples of approximately 2.0 cm × 2.0 cm were obtained from the medial area of the left leg under sterile conditions. The tissue samples were quickly transferred to pre-chilled PBS buffer (0.01 mol / L, pH 7.4) containing double-antibiotics (penicillin 100 IU / mL, streptomycin 100 μg / mL). After rinsing three times, the subcutaneous fat layer and epidermal tissue were removed. The dermal tissue was placed in a sterile culture dish and finely cut into approximately 1 mm thick slices using ophthalmic scissors. 3The tissue fragments were treated with a stepwise enzymatic digestion method using 0.2% type I collagenase (digested at 37°C for 2h) combined with 0.25% trypsin (digested at 37°C for 15min). The cell suspension was collected after filtration through a 100μm cell sieve. The cell suspension was centrifuged at 4°C and 1000rpm for 10min. After discarding the enzymatic solution, the cells were resuspended in DMEM complete medium containing 10% fetal bovine serum. After determining the cell density using a hemocytometer, 5×10 4 cells / cm 2 Transfer the cell suspension to a cell culture flask at a seeding density of 100 μg / mL and place in a cell culture incubator at 37°C and 5% CO2 for primary culture. Observe cell adhesion daily under a microscope. When cell confluence reaches 80-90%, subculture using 0.25% trypsin.

[0057] 1.3 CCK-8 assay to detect the effects of various drugs on canine fibroblast proliferation

[0058] Canine fibroblasts in the logarithmic growth phase were cultured at a rate of 1×10 5 Cells were seeded into 96-well plates at a density of 100 μL per well. A blank control group and experimental groups 1-5 were set up with 3 replicates per group. The cells were cultured overnight at 37°C in a 5% CO2 environment. After the cells attached to the wall, 200 μL of culture medium was given to the blank control group, and 200 μL of culture medium containing the drug stock solution of each experimental group (final concentration of 50 μg / mL) was given to the experimental groups 1-5 respectively. After further culture for 24 hours, 10 μL of CCK-8 was added to each well and incubated for 3 hours. The absorbance at 450 nm was measured using a microplate reader to calculate the cell viability. Cell viability = (OD 药物组 -OD 对照组 ) / OD 对照组 ×100%.

[0059] 1.4SA-β-galactosidase activity assay

[0060] Canine fibroblasts in the logarithmic growth phase were cultured at a rate of 1×10 5Cells were seeded at a density of 100 μL per well in a 96-well plate at 400 μg / mL and cultured in a 37°C, 5% CO2 incubator for 24 hours. After cell attachment, they were randomly divided into seven groups: a blank control group, a model control group, and experimental groups 1-5, with three replicates per group. Except for the blank control group, cells in all other groups were treated with 50 μL of 10 g / L D-galactose to induce senescence. Simultaneously, the cells were treated with the drug. Experimental groups 1-5 were each given 50 μL of culture medium containing the respective drug solution (final concentration 50 μg / mL), while the blank control group was supplemented with an equal volume of complete DMEM medium. All treatment groups and the model control group were co-cultured under the same culture conditions for 72 hours. The SA-β-galactosidase staining kit was used for detection. Five fields of view were randomly selected under a light microscope (200× magnification), and the percentage of blue-stained cells was counted. The positive cell rate was calculated according to the following formula: positive cell rate = (number of positive cells / total number of cells) × 100%.

[0061] 1.5 Antioxidant index detection

[0062] Canine fibroblasts in the logarithmic growth phase were cultured at a rate of 1×10 5 Cells were seeded at a density of 100 μL per well in a 96-well plate at 100 μL per well and incubated at 37°C in a 5% CO2 incubator for 24 hours. After cell attachment, they were randomly divided into seven groups: a blank control group, a model control group, and experimental groups 1-5, with three replicates per group. Except for the blank control group, cells in all other groups were treated with 50 μL of 10 g / L D-galactose to induce senescence. Simultaneously, the drug treatments were administered to experimental groups 1-5. Experimental groups 1-5 were each given 50 μL of culture medium containing the respective drug solution (final concentration 50 μg / mL), while the blank control group was supplemented with an equal volume of complete DMEM medium. All treatment groups and the model control group were co-cultured under the same conditions for 72 hours. After incubation, the supernatant was collected and assayed for superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities and malondialdehyde (MDA) content according to the ELISA kit instructions.

[0063] 1.6 Statistical methods

[0064] Data were analyzed using Graphpad Prism 8 statistical software, and the mean The data are expressed as ± standard deviation (s). Oneway ANOVA was used for statistical analysis, and the differences were considered statistically significant when P < 0.05.

[0065] 2 Results

[0066] 2.1 Effects of various drugs on canine fibroblast cell viability

[0067] Table 1 shows the comparison of canine fibroblast viability in each group. Compared with the blank control group, the cell viability of canine fibroblasts in test groups 1-5 showed no significant difference (P>0.05), indicating that each group of drugs had no significant toxic effect on canine fibroblasts and had good safety.

[0068] Table 1 Comparison of cell viability in each group

[0069] Grouping Cell viability (%) Blank control group 100.00±0.00 Experimental group 1 101.58±3.65 Experimental Group 2 96.09±3.27 Experiment 3 groups 102.10±1.96 Experiment 4 groups 99.02±3.70 Experiment 5 groups 98.49±3.13

[0070] 2.2 Effects of various drugs on the positive cell rate of SA-β-galactosidase staining in canine fibroblasts

[0071] As shown in Table 2, compared with the blank control group, the SA-β-galactosidase staining-positive cell rate in the model control group was significantly increased (P<0.01), indicating that the cell aging model was successfully constructed; compared with the model control group, the SA-β-galactosidase staining-positive cell rate in test groups 1 to 5 decreased to varying degrees, among which the decrease in test group 1 was the most significant, indicating that the additive of the present invention can effectively inhibit the expression of SA-β-gal, a cell aging marker, and has significant anti-aging efficacy.

[0072] Table 2 Comparison of SA-β-galactosidase staining positive cell rates in each group

[0073] Grouping Positive cell rate (%) Blank control group 10.49±1.31 Model control group <![CDATA[31.88±3.17 ## ]]> Experimental group 1 <![CDATA[15.71±2.58 ** ]]> Experimental Group 2 28.70±1.89 Experiment 3 groups <![CDATA[24.95±2.34 * ]]> Experiment 4 groups <![CDATA[18.94±3.12 ** ]]> Experiment 5 groups <![CDATA[23.74±2.37 * ]]>

[0074] Note: Compared with the blank control group, # P<0.05, ## P<0.01; compared with the model control group, * P<0.05, ** P<0.01.

[0075] 2.3 Effects of various drugs on antioxidant indices of canine fibroblasts

[0076] Figure 1 The results showed a comparison of the antioxidant indices of canine fibroblasts in each group. Compared with the blank control group, the SOD and GSH-Px activities of the model control group were significantly reduced (P<0.01), while the MDA content was significantly increased (P<0.01). Compared with the model control group, the SOD and GSH-Px activities of test groups 1-5 increased to varying degrees, while the MDA content was significantly reduced. Among them, the effect of test group 1 in enhancing the activity of antioxidant enzymes and reducing the MDA content was the most significant (P<0.01), indicating that the additive of the present invention may exert its anti-aging effect by enhancing the activity of antioxidant enzymes and reducing the accumulation of lipid peroxidation products.

[0077] 3 Conclusion

[0078] This study revealed the synergistic anti-aging mechanism of the composite system of proanthocyanidins, pseudo-Nannochloropsis oil, Chinese herbal extracts and maqui berry anthocyanins. The experimental data showed that compared with the experimental group 2 (without proanthocyanidins and pseudo-Nannochloropsis oil), the SA-β-galactosidase staining positive cell rate of canine fibroblasts in the experimental group 1 (complete formula group) was significantly reduced by 12.99%, while the activities of key enzymes SOD and GSH-Px in the antioxidant defense system increased by 44.31% and 60.07% respectively, and the content of lipid peroxidation product MDA decreased by 37.45%, indicating that proanthocyanidins and pseudo-Nannochloropsis oil have a synergistic effect in antioxidant defense and delaying cell aging; on the other hand, compared with the experimental group 4 (without maqui berry anthocyanins), SA-β-g The SA-β-gal positive cell rate decreased by 3.23%, while the SOD and GSH-Px activities increased by 17.76% and 17.77%, respectively, and the MDA content decreased significantly by 33.45%, indicating that the addition of maqui berry anthocyanins made an important contribution to the overall anti-aging effect; in addition, mistletoe and locust in Chinese herbal extracts also showed unique anti-aging activity. Compared with the experimental group 5 (excluding mistletoe and locust), the SA-β-gal positive cell rate in the experimental group 1 decreased by 8.03%, while the SOD and GSH-Px activities increased by 4.8% and 11.75%, respectively, and the MDA content decreased significantly by 19.37%.

[0079] Comprehensive analysis found that test group 1 (i.e., the complete formula group) showed the best anti-aging effect in all test indicators, confirming that the additive of the present invention, by constructing a multi-component synergistic network, jointly constructed a dual anti-aging system based on antioxidant defense and aging marker regulation. Its mechanism of action is mainly reflected in three aspects: significantly inhibiting the expression of aging-related β-galactosidase biomarkers, strongly activating the endogenous antioxidant enzyme system, and effectively blocking the lipid peroxidation chain reaction, providing a theoretical basis and scientific foundation for subsequent in vivo pharmacodynamic evaluation.

[0080] Test Example 2

[0081] 1 Materials and Methods

[0082] 1.1 Experimental dogs

[0083] Twenty-four healthy elderly beagle dogs aged 9-12 years, half male and half female, were purchased from Changzhou Belle Experimental Animal Breeding Co., Ltd.

[0084] 1.2 Trial Drugs

[0085] The additives used in the microencapsulated preparation group were prepared according to the method described in Example 1.

[0086] The additives used in the common dosage form group were prepared according to the method described in Comparative Example 1.

[0087] 1.3 Treatment

[0088] Twenty-four beagle dogs were randomly divided into three groups: control group, microencapsulated preparation group, and conventional preparation group, with 8 dogs in each group. The blank control group was fed with complete dog food daily, while the microencapsulated preparation group and conventional preparation group were fed with complete dog food containing 2% additives twice a day for 8 consecutive weeks.

[0089] 1.4 Detection indicators

[0090] 1.4.1 Cognitive testing

[0091] A modified Y-maze evaluation system was used. The dogs were first acclimated to the maze environment for 2 days, and then entered the learning phase of training. The latency and number of errors for each experimental dog to find the fixed target area from the starting point were recorded. This was repeated several times daily until the standard was reached (latency ≤ 60 seconds and number of errors ≤ 1 in three consecutive tests). Memory retention tests were then conducted 1 hour and 24 hours after the end of training. The difference between short-term and long-term memory was assessed by comparing the latency and number of errors in the two tests.

[0092] 1.4.2 Canine Cognitive Dysfunction Rating Scale (CCDR)

[0093] The CCDR scale (Table 3) was used to evaluate the changes in cognitive dysfunction in each group of experimental dogs based on the following 13 items. The total score ranged from 0 to 80 points, specifically divided into: 0-39 points for normal cognitive function, 40-49 points for risk of cognitive decline, and 50-80 points for diagnosis of cognitive dysfunction.

[0094] Table 3 Canine Cognitive Dysfunction Rating Scale

[0095]

[0096]

[0097] 1.4.3 Plasma NAD+ Detection

[0098] The dog was restrained in a lateral position, the hair of the jugular vein was removed, and venous puncture was performed using a 22G pillow connected to an EDTA anticoagulant tube after iodine disinfection. 5 mL of whole blood was slowly drawn and immediately mixed by gentle inversion and anticoagulation. The blood was centrifuged at 4°C and 3000 rpm for 10 min, and the upper light yellow plasma was collected. Nicotinamide adenine dinucleotide (NAD + )level.

[0099] 1.4.4 Detection of inflammatory factors in synovial fluid

[0100] After the experiment, the dog was restrained in a lateral recumbent position, the hair around the knee joint was removed, and the area was disinfected with iodine followed by alcohol deiodination. The surgical site was then sterilely isolated. The joint space was palpated, and a 22G pillow connected to a sterile syringe was rapidly inserted perpendicularly through the skin. After penetrating the joint capsule, the syringe was slowly withdrawn until viscous synovial fluid began to flow in. After the needle was removed, blood was withdrawn by pressure. The aspirated fluid was immediately transferred to an EDTA anticoagulant tube and centrifuged at 3000 rpm for 10 minutes at 4°C. The supernatant was collected and the levels of inflammatory factors such as TNF-α, IL-1β, and IL-6 in the knee joint synovial fluid were measured according to the ELISA kit instructions.

[0101] 1.5 Statistical methods

[0102] Data were analyzed using Graphpad Prism 8 statistical software, and the mean The data are expressed as ± standard deviation (s). Oneway ANOVA was used for statistical analysis, and the differences were considered statistically significant when P < 0.05.

[0103] 2 Results

[0104] 2.1 Impact on cognitive function

[0105] As shown in Table 4, compared with the control group, the latency of the test dogs in each drug-treated group was significantly shortened 1 hour and 24 hours after the end of training (P<0.05), and the number of errors was also significantly reduced (P<0.05). Among them, the latency and number of errors of the test dogs in the microencapsulated preparation group were significantly lower than those in the conventional preparation group, indicating that the additive of the present invention can effectively improve the cognitive function of the test dogs, thereby exerting an anti-aging effect.

[0106] Table 4 Comparison of cognitive function of experimental dogs in each group

[0107]

[0108] Note: Compared with the control group, * P<0.05, ** P<0.01.

[0109] 2.2 Impact on CCDR score

[0110] As shown in Table 5, compared with the control group, the CCDR scores of the test dogs in each drug-treated group showed a downward trend (P<0.05 or P<0.01), among which the CCDR score of the microencapsulated preparation group was significantly lower than that of the ordinary preparation group (P<0.05), indicating that the additive of the present invention can significantly improve the cognitive function of the test dogs and has a clear intervention effect on delaying cognitive dysfunction in pets.

[0111] Table 5 Comparison of CCDR scores of experimental dogs in each group

[0112] Grouping CCDR score control group <![CDATA[52.24±4.68 a ]]> Microencapsulated preparation group <![CDATA[34.34±3.15 b ]]> Ordinary preparation group <![CDATA[43.68±2.79 a ]]>

[0113] 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).

[0114] 2.3 Effects of plasma NAD + The impact of level

[0115] As shown in Table 6, compared with the control group, the plasma NAD + The levels showed a significant upward trend (P<0.05 or P<0.01), among which the plasma NAD + The level was significantly higher than that of the common preparation group (P<0.05), indicating that the additive of the present invention can effectively increase the plasma NAD of the experimental dogs. + levels, thereby delaying the aging process of pets.

[0116] Table 6 Plasma NAD of each group of experimental dogs + Level comparison (μmol / L)

[0117] Grouping <![CDATA[NAD + Level]]> control group <![CDATA[1.10±0.16 c ]]> Microencapsulated preparation group <![CDATA[1.48±0.13 a ]]> Ordinary preparation group <![CDATA[1.40±0.06 b ]]>

[0118] 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).

[0119] 2.4 Effects on the levels of inflammatory factors in synovial fluid

[0120] As shown in Table 7, compared with the control group, the levels of inflammatory factors (TNF-α, IL-1β, IL-6) in the synovial fluid of the test dogs in each drug-treated group showed a downward trend (P<0.05). Among them, the levels of TNF-α, IL-1β, and IL-6 in the synovial fluid of the test dogs in the microencapsulated preparation group were significantly lower than those in the conventional preparation group (P<0.05), indicating that the additive of the present invention can effectively inhibit the release of inflammatory factors, reduce the body's inflammatory response, and thus delay the aging process.

[0121] Table 7 Comparison of inflammatory factor levels in the synovial fluid of the experimental dogs in each group (pg / mL)

[0122] Grouping TNF-α IL-1β IL-6 control group <![CDATA[203.86±8.42 a ]]> <![CDATA[184.76±8.98 a ]]> <![CDATA[255.92±8.98 a ]]> Microencapsulated preparation group <![CDATA[154.67±7.73 c ]]> <![CDATA[110.55±13.30 c ]]> <![CDATA[204.21±11.74 c ]]> Ordinary preparation group <![CDATA[179.31±6.71 b ]]> <![CDATA[142.26±9.45 b ]]> <![CDATA[230.06±8.82 b ]]>

[0123] 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).

[0124] 3 Conclusion

[0125] The above test results show that the additive of the present invention can significantly improve the cognitive function of the experimental dogs, effectively reduce their cognitive dysfunction rating scale (CCDR) scores, and thus delay the progression of cognitive dysfunction; in addition, the additive can also effectively increase the plasma nicotinamide adenine dinucleotide (NAD + ) levels and reduced levels of inflammatory factors such as TNF-α, IL-1β, and IL-6 in synovial fluid, thereby exerting anti-aging effects in multiple dimensions. The microencapsulated formulation group showed significantly better improvements in each of these indicators than the conventional formulation group (P<0.05), demonstrating that microencapsulation technology can significantly enhance the bioavailability and anti-aging efficacy of additives, providing a safe and effective new solution for delaying aging in pets.

[0126] 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 pet anti-aging additive based on composite functional ingredients, characterized in that: The additive is prepared from the following active ingredients in percentage by weight: 20-35 parts of pseudo-nanochloridia oil, 15-30 parts of proanthocyanidins, 10-25 parts of arabinoxylan, 5-20 parts of N-acetylglucosamine, 12 parts of Bifidobacterium longum M-633, 6-25 parts of nano-traditional Chinese medicine extracts, and 1-10 parts of maqui berry anthocyanins.

2. The pet anti-aging additive based on composite functional ingredients according to claim 1, characterized in that: The additive is prepared from the following active ingredients in percentage by weight: 25-30 parts of pseudo-nanochloridia oil, 20-25 parts of proanthocyanidins, 15-20 parts of arabinoxylan, 10-15 parts of N-acetylglucosamine, 8 parts of Bifidobacterium longum M-635, 10-20 parts of nano-traditional Chinese medicine extract, and 2-7 parts of maqui berry anthocyanins.

3. The pet anti-aging additive based on composite functional ingredients according to claim 1, characterized in that: The additive is prepared from the following active ingredients in percentage by weight: 25 parts of pseudo-nanochloridia oil, 20 parts of proanthocyanidins, 15 parts of arabinoxylan, 12 parts of N-acetylglucosamine, 37 parts of Bifidobacterium longum M-63, 15 parts of nano-traditional Chinese medicine extract, and 3 parts of maqui berry anthocyanins.

4. A pet anti-aging additive based on composite functional ingredients according to any one of claims 1 to 3, characterized in that: The nano traditional Chinese medicine extract is compounded from the following raw materials according to the mass ratio: frankincense: yam: mistletoe: locust=3-10:2-8:1-3:1-3.

5. The pet anti-aging additive based on composite functional ingredients according to any one of claims 1 to 3, characterized in that: The nano-TCM extract is compounded from the following raw materials in a mass ratio of: frankincense: yam: mistletoe: locust=7:5:2:

1.

6. The method for preparing a pet anti-aging additive based on composite functional ingredients according to any one of claims 1 to 5, characterized in that: The following steps are involved: (1) Preparation of nano-TCM extracts: Frankincense, Chinese yam, mistletoe and locust were weighed separately, cleaned and pulverized into fine powder; 70% ethanol solution was used as the extraction medium for ultrasonic extraction and then vacuum rotary evaporation was used to obtain the extract; the extract was dispersed in olive oil at a mass ratio of 1:5 to form an oil phase, and 2% Tween-80 was dissolved in water as the aqueous phase, and the nanoemulsion was prepared by high-speed shear emulsification and high-pressure homogenization. (2) Composite liposome encapsulation: Soybean lecithin and cholesterol were mixed in a 2:1 molar ratio, dissolved in methanol, and subjected to a rotary evaporator to form a uniform lipid film; the lipid film was re-dissolved in PBS buffer, and proanthocyanidins and maqui berry anthocyanins were added. After ultrasonic hydration at 37°C for 30-60 min, nanoliposomes were obtained by extrusion through a 0.1 μm polycarbonate membrane. (3) After homogenizing the above-mentioned nano-TCM extract, nanoliposomes and pseudomicrocystis oil, arabinoxylan and N-acetylglucosamine were added, and microencapsulated by low-temperature spray drying. Subsequently, the additive of the present invention was obtained by mixing with Bifidobacterium longum M-63 freeze-dried powder under sterile conditions.

7. The method for preparing a pet anti-aging additive based on composite functional ingredients according to claim 6, characterized in that: In the step (1), the power of ultrasonic extraction is 40kHz, the temperature is 40-55°C, the time is 20-30min, the speed of high-speed shear emulsification is 10000rpm, the time is 5-15min, and the pressure of the high-pressure homogenizer is 100MPa; the process parameters of low-temperature spray drying in step (3) are an inlet air temperature of 120°C, an outlet air temperature of 60°C, and an atomization pressure of 0.3MPa.

8. Use of the additive according to any one of claims 1 to 5 in the preparation of a product for delaying pet aging.

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