Composition for promoting lactation performance of pets as well as preparation method and application of composition

Abstract

The invention provides a composition for promoting the lactation performance of pets and a preparation method and application thereof, and belongs to the technical field of animal health care product preparation. Comprising a composition capable of promoting lactation, a perilla seed extract, alfalfa flavone, eucommia ulmoides chlorogenic acid and other raw materials, and further comprises one or more of butyric acid, malic acid, disodium 5 '-guanylate, benzoic acid, tartaric acid, palm fatty acid powder, nicotinamide, cystine, leucine, tyrosine, lecithin, linolenic acid and linoleic acid. According to the composition for promoting the lactation function of the pets and the preparation method of the composition, the composition has a synergistic effect on promoting the lactation function, the cell viability of epithelial cells of the mammary glands of dogs can be improved, and the contents of lactose, lactoprotein, milk fat and the like in the cells are increased. The health-care agent is prepared by mixing the perilla seed extract, the alfalfa flavone and the eucommia ulmoides chlorogenic acid according to a certain proportion and adding the additive, so that the lactation function of pets is promoted, and the problems of insufficient breast milk and the like are solved.

Description

Technical Field

[0001] The invention belongs to the technical field of preparation of animal health products, and in particular relates to a composition for promoting lactation function of pets and a preparation method thereof. Background Art

[0002] For newborn dogs and cats, breast milk is a very good nutrient, especially colostrum, which contains a large amount of maternal antibodies. Due to the physical condition of the pets themselves or the large litter size, some dogs and cats will have insufficient breast milk, resulting in a low survival rate of puppies. The main reasons for insufficient breast milk in dogs and cats include malnutrition, physical fatigue, incorrect breastfeeding posture and genetic problems. Malnutrition is one of the main reasons for insufficient breast milk. The solutions to insufficient breast milk in dogs and cats include the following aspects: 1. Increase nutritional intake 2. Use Chinese herbal medicine 3. Maintain good living habits 4. Correct breastfeeding posture 5. Artificial feeding.

[0003] Alfalfa, Eucommia ulmoides and Perilla seeds are plants with milk-increasing functions, and have certain applications in treating lack of milk and increasing animal milk production. Feeding alfalfa can improve the growth performance, lactation performance and feed conversion rate of ruminants. Alfalfa contains a variety of active ingredients such as polysaccharides, saponins and flavonoids, among which flavonoids have the highest content. Flavonoids have the properties of anti-pathogenic microorganisms, immunostimulation and antioxidant, so they can be widely used as a feed additive in animal production. The appropriate addition of alfalfa flavonoids can increase the feed intake of dairy cows and regulate the body's hormone secretion and immune performance. In addition, Eucommia ulmoides extract contains a variety of active pharmaceutical ingredients such as flavonoids, polysaccharides, cyclopentane ether terpenes, phenylpropanoids, lignans, etc. The active substances contained can promote animal growth, improve its production performance, enhance animal immunity, and have anti-stress effects, and are widely used. Adding different levels of Eucommia ulmoides extract can improve the reproductive performance, lactation performance of pregnant sows and the growth performance of offspring piglets. Perilla plants have antibacterial, antiviral, anti-inflammatory, anti-allergic and antioxidant effects. Studies have shown that perilla can significantly increase the milk production of dairy cows.

[0004] At present, there are some products on the market that promote animal lactation. CN108208370A discloses a feed additive that uses perilla to improve the immunity of dairy cows during the lactation period and its preparation method. Specifically, the extracts of perilla leaves, dandelions, honeysuckle, Shuanghuanglian, forsythia and isatis root can effectively promote milk secretion, improve milk production and quality. CN107873978A discloses a feed for improving the milk production of dairy cows. The formula includes: 14-22 parts of corn protein powder, 3-6 parts of soybean lecithin, 4-7 parts of brewer's yeast, 2-4 parts of rosemary powder, 5-12 parts of black bean powder, 5-8 parts of Chinese yam, 2-7 parts of gracilaria, 2-8 parts of kudzu root, 3-6 parts of wolfberry, 5-9 parts of peanut, 6-10 parts of alfalfa, 2-8 parts of hawthorn, 3-8 parts of acanthopanax bark, 1-6 parts of poplar flower, 4-10 parts of Viola yedoensis, 5-10 parts of plantain, 2-7 parts of licorice, 3-7 parts of kumquat, 4-11 parts of dendrobium, 4-9 parts of platycodon, 2-5 parts of polygonatum, 4-8 parts of red dates, 3-5 parts of perilla and 0.3-0.5 parts of a complex vitamin additive. CN110403084A discloses a Chinese medicine composite peptide for improving the milk production and milk quality of dairy cows, and its preparation method and application. The Chinese medicine composite peptide is composed of soybean peptide, astragalus hydrolysate, angelica hydrolysate, fenugreek hydrolysate, Eucommia leaf hydrolysate, loofah hydrolysate and vitamins. CN108813154A discloses the application of Eucommia leaf extract in the preparation of additives for improving the breeding performance and lactation of sows. Feeding sows with Eucommia leaf extract can improve follicle secretion, promote embryonic development; increase litter size by 5%; increase lactation by 10%; and advance estrus by 3 to 10 days. The above patents are mainly used in farmed animals such as dairy cows and pigs. There are no lactation products for companion animals such as dogs and cats, and the existing patents have not proposed the mechanism of functional ingredients promoting pet lactation or the key biological pathways affected.

[0005] In view of the lack of lactation products for dogs and cats on the market, this paper proposes a new strategy to promote lactation for dogs and cats based on precise nutritional metabolite regulation. Through in-depth research on the metabolic mechanism of lactation, we have discovered multiple key biological metabolic pathways. The metabolite regulation of these pathways provides a more comprehensive solution for promoting lactation in dogs and cats, and has developed a new formula. Summary of the invention

[0006] In view of this, the present invention aims to provide a composition for promoting lactation function of pets and a preparation method thereof, wherein the composition can increase the content of lactose, milk protein and milk fat in canine mammary gland cells.

[0007] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0008] A composition for promoting lactation function of pets, comprising perilla seed extract, alfalfa flavonoids and eucommia chlorogenic acid, wherein the weight proportions are:

[0009] 3 to 60 parts of perilla seed extract, 3 to 60 parts of alfalfa flavonoids, and 3 to 60 parts of eucommia chlorogenic acid;

[0010] The preferred weight proportions are: 60 parts of perilla seed extract, 3 parts of alfalfa flavonoids, and 3 parts of eucommia chlorogenic acid.

[0011] The health-care agent for promoting lactation function of pets of the present invention is composed of the following raw materials in parts by weight:

[0012] 3-60 parts of perilla seed extract, 3-60 parts of alfalfa flavonoids, 3-60 parts of eucommia chlorogenic acid, 1-2 parts of butyric acid, 1-2 parts of malic acid, 0.5-1 parts of disodium 5'-guanylate, 0.5-1 parts of benzoic acid, 1-2 parts of tartaric acid, 5-10 parts of palm fatty acid powder, 3-5 parts of niacinamide, 0.1-0.2 parts of cystine, 0.1-0.2 parts of leucine, 0.1-0.2 parts of tyrosine, 1-2 parts of lecithin, 4-6 parts of linolenic acid, and 2-5 parts of linoleic acid.

[0013] The preferred raw materials are the following parts by weight:

[0014] 60 parts of perilla seed extract, 3 parts of alfalfa flavonoids, 3 parts of eucommia chlorogenic acid, 2 parts of butyric acid, 2 parts of malic acid, 1 part of disodium 5'-guanylate, 1 part of benzoic acid, 2 parts of tartaric acid, 10 parts of palm fatty acid powder, 5 parts of niacinamide, 0.2 parts of cystine, 0.2 parts of leucine, 0.2 parts of tyrosine, 2 parts of lecithin, 6 parts of linolenic acid, and 5 parts of linoleic acid.

[0015] The perilla seed extract of the present invention is prepared according to the following steps:

[0016] Step (1): weigh perilla seeds, crush them, add 60% ethanol, extract them under reflux at 55° C. for 2 times, each time for 1 hour, filter, and obtain a perilla seed extract; wherein the mass to volume ratio of perilla seeds to 60% ethanol is 1:10 (g / mL).

[0017] Step (2): The perilla seed extract is concentrated to dryness under reduced pressure using a vacuum concentrator pump to obtain a perilla seed extract.

[0018] The alfalfa flavonoids of the present invention are prepared according to the following steps:

[0019] Step (1): fresh alfalfa is washed, dried and crushed into powder, an appropriate amount of sodium hydroxide solution is added, and reflux extraction is performed in a constant temperature water bath at 50° C. for 2 hours, and filtered to obtain an extract; wherein the mass to volume ratio of dry alfalfa to sodium hydroxide solution is 1:10 (g / mL);

[0020] Step (2): adding 60% ethanol solution to the extract, extracting at 80° C. for 1 hour, and centrifuging and drying to obtain alfalfa flavonoids; wherein the volume ratio of the extract to the 60% ethanol is 1:10 (mL / mL);

[0021] The Eucommia ulmoides chlorogenic acid of the present invention is prepared according to the following steps:

[0022] Step (1): crushing the Eucommia leaves, adding 70% ethanol, extracting at 60° C. for 3 times, each time for 1 hour, filtering to obtain an extract; wherein the mass to volume ratio of the Eucommia leaves to the 70% ethanol is 1:30 (g / mL);

[0023] Step (2): The extract is concentrated to dryness using a vacuum concentration and pressure reduction pump to obtain Eucommia ulmoides chlorogenic acid.

[0024] The method for preparing the health-care agent for promoting the lactation function of pets of the present invention is characterized by following the following steps:

[0025] Step (1): weighing 3-60 parts of perilla seed extract, 3-60 parts of alfalfa flavonoids, and 3-60 parts of eucommia chlorogenic acid;

[0026] Step (2): Mix the Chinese herbal medicine weighed in step (1) with the additives, and add water until the mixed raw materials are submerged;

[0027] Step (3): After stirring evenly, granulation and air drying are performed to obtain a granular health-care agent.

[0028] Preferably, the additive in step (2) is one or more of butyric acid, malic acid, disodium 5'-guanylate, benzoic acid, tartaric acid, palm fatty acid powder, nicotinamide, cystine, leucine, tyrosine, lecithin, linolenic acid and linoleic acid.

[0029] Preferably, the additives in step (2) are 1-2 parts of butyric acid, 1-2 parts of malic acid, 0.5-1 parts of disodium 5'-guanylate, 0.5-1 parts of benzoic acid, 1-2 parts of tartaric acid, 5-10 parts of palm fatty acid powder, 3-5 parts of niacinamide, 0.1-0.2 parts of cystine, 0.1-0.2 parts of leucine, 0.1-0.2 parts of tyrosine, 1-2 parts of lecithin, 4-6 parts of linolenic acid, and 2-5 parts of linoleic acid.

[0030] The application of the perilla seed extract, alfalfa flavonoids and eucommia chlorogenic acid of the present invention can significantly promote the proliferation activity of canine mammary epithelial cells.

[0031] The application of the perilla seed extract, alfalfa flavonoids and eucommia chlorogenic acid of the present invention can significantly increase the intracellular lactose, milk protein and milk fat content.

[0032] Compared with the prior art, the composition for promoting lactation function of pets and the preparation method thereof of the present invention have the following beneficial effects:

[0033] 1. The present invention studies the cellular level of dogs and cats, and on this basis, deeply explores the metabolic mechanism of dog and cat lactation, and finds the effects on amino acid metabolism, fatty acid biosynthesis, glycerophospholipid metabolism, etc. By introducing key ingredients such as butyric acid, malic acid, disodium 5'-guanylate, benzoic acid, tartaric acid, palm fatty acid powder, nicotinamide, cystine, leucine, tyrosine, lecithin, linolenic acid and linoleic acid, it is more conducive to solving the problem of insufficient breast milk for dogs and cats.

[0034] 2. The composition of the present invention effectively regulates key biochemical pathways in the lactation process by supplementing a variety of nutritional metabolic regulators, maintains cell membrane stability, promotes cell metabolism and repair, and provides energy for cells. This comprehensive nutritional support method accurately optimizes and enhances the effects of traditional methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0036] Figure 1 This is the effect of the perilla seed extract, alfalfa flavonoids and eucommia chlorogenic acid in the present invention on the viability of canine mammary epithelial cells. BC in the figure represents the untreated group, and 1-320 in the abscissa represents that the concentration range of the perilla seed extract, alfalfa flavonoids or eucommia chlorogenic acid is 1-320 μg / mL.

[0037] Figure 2 This is the effect of the combination of perilla seed extract, alfalfa flavonoids and eucommia chlorogenic acid in the present invention on the concentrations of lactose (A) and lactase (B). In the figure, BC represents the untreated group, PSE represents the perilla seed extract treatment group, CGA represents the eucommia chlorogenic acid treatment group, AFF represents the alfalfa flavonoids treatment group, and PSE:CGA:AFF represents perilla seed extract: eucommia chlorogenic acid: alfalfa flavonoids.

[0038] Figure 3 This is the effect of the combination of perilla seed extract, alfalfa flavonoids and eucommia chlorogenic acid in the present invention on the concentrations of triglycerides (A) and fatty acid synthase (B). In the figure, BC represents the untreated group, PSE represents the perilla seed extract treatment group, CGA represents the eucommia chlorogenic acid treatment group, AFF represents the alfalfa flavonoids treatment group, and PSE:CGA:AFF represents perilla seed extract: eucommia chlorogenic acid: alfalfa flavonoids.

[0039] Figure 4This is the effect of the combination of perilla seed extract, alfalfa flavonoids and eucommia chlorogenic acid in the present invention on the concentrations of α-lactalbumin (A) and β-casein (B). In the figure, BC represents the untreated group, PSE represents the perilla seed extract treated group, CGA represents the eucommia chlorogenic acid treated group, AFF represents the alfalfa flavonoids treated group, and PSE:CGA:AFF represents perilla seed extract: eucommia chlorogenic acid: alfalfa flavonoids.

[0040] Figure 5 This is the effect of the combination of perilla seed extract, alfalfa flavonoids and eucommia chlorogenic acid in the present invention on the concentrations of progesterone receptor (A) and prolactin receptor (B). In the figure, BC represents the untreated group, PSE represents the perilla seed extract treatment group, CGA represents the eucommia chlorogenic acid treatment group, AFF represents the alfalfa flavonoids treatment group, and PSE:CGA:AFF represents perilla seed extract: eucommia chlorogenic acid: alfalfa flavonoids.

[0041] Figure 6 It is a volcano plot of differential metabolites between the groups in the present invention, in which (A) is a volcano plot of differential metabolites before and after treatment with perilla seed extract; (B) is a volcano plot of differential metabolites before and after treatment with alfalfa flavonoids; (C) is a volcano plot of differential metabolites before and after treatment with eucommia chlorogenic acid; P-value refers to the statistical significance of the t-test; Fold Change refers to the quantitative ratio of two groups of experimental substances; VIP refers to variable projection importance.

[0042] Figure 7 It is a cluster heat map of differential metabolites among the groups in the present invention. DETAILED DESCRIPTION

[0043] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0044] The present invention will be described in detail below with reference to the accompanying drawings and in conjunction with embodiments.

[0045] The preparation method of the health-care agent for promoting the lactation function of pets of the present invention comprises the following steps:

[0046] Step (1): weigh 100 g of perilla seeds, crush them, add 1000 mL of 60% ethanol, reflux extract at 55° C. for 2 times, each time for 1 hour, filter, and obtain a perilla seed extract;

[0047] Step (2): The perilla seed extract was concentrated to dryness under reduced pressure using a vacuum concentrator pump to obtain 10 g of perilla seed extract.

[0048] The preparation method of the health-care agent for promoting the lactation function of pets of the present invention comprises the following steps:

[0049] Step (1): Wash and dry fresh alfalfa and grind it into 100 g of powder, add 1000 mL of 0.1 mol / L sodium hydroxide solution, reflux extract in a constant temperature water bath at 50° C. for 2 hours, filter, and obtain 200 mL of extract;

[0050] Step (2): add 2000 mL of 60% ethanol solution to the extract, extract at 80° C. for 1 hour, and centrifuge and dry to obtain 8 g of alfalfa flavonoids.

[0051] The preparation method of the health-care agent for promoting the lactation function of pets of the present invention, wherein the eucommia chlorogenic acid is prepared by the following steps:

[0052] Step (1): 100 g of Eucommia ulmoides leaves were crushed, and then added with 3000 mL of 70% ethanol, and extracted at 60° C. for 3 times, each time for 1 hour, and filtered to obtain an extract;

[0053] Step (2): The extract was concentrated to dryness using a vacuum concentration and decompression pump to obtain 8 g of Eucommia ulmoides chlorogenic acid. I. Effect test of the composition on canine mammary epithelial cells

[0054] 1. Test methods

[0055] (1) Cell culture

[0056] PETCC2526 canine mammary epithelial cells were cultured in M-3 medium containing 10% fetal bovine serum in a 37°C, 5% CO2 incubator. When the cells grew to 80% confluence, cell passage and experiment were performed.

[0057] (2) Cell viability assay

[0058] Canine mammary cells were digested and collected with trypsin containing 0.25% EDTA, and the cell concentration was adjusted to 8×104 / mL. They were inoculated into 96-well cells, and PBS was added around them for 24 hours. 100 μL of different concentrations of perilla seed extract, alfalfa flavonoids, and eucommia chlorogenic acid (1, 5, 10, 20, 40, 80, 160, 320 μg / mL) were added as the experimental group, and no treatment was performed as the control group (BC). Each group was repeated 3 wells and cultured for 24 hours at 37°C and 5% CO2. 10 μL of CCK-8 solution was added to each well, and after incubation for 0.5-1h, the absorbance value of each well at a wavelength of 450nm was detected using an enzyme reader to calculate the cell viability.

[0059] Cell viability (%) = (A test group - A blank) / (A control group - A blank) × 100%

[0060] (3) Lactose and lactase content detection

[0061] The groups were set up as control group (BC) and experimental group (20 μg / mL perilla seed extract, 20 μg / mL alfalfa flavonoids, 20 μg / mL eucommia chlorogenic acid, mass ratio of perilla seed: alfalfa flavonoids: eucommia chlorogenic acid = (1-20): (1-20): (1-20)). 2×105 cells / mL of cell suspension were inoculated in 6-well plates, 2 mL per well, and placed in a CO2 incubator for 24 h. Subsequently, 2 mL of different groups of drug solutions were added for intervention for 24 h, and each group was repeated 3 times. The cell supernatant was then collected for lactose and lactase determination. The specific operation was carried out according to the instructions of the detection kit, and the absorbance value at a wavelength of 540 nm was measured using an enzyme marker.

[0062] (4) Milk fat content detection

[0063] The cell treatment method is the same as (3). The cell supernatant was collected and used for triglyceride and fatty acid synthase determination. The specific operation was carried out according to the instructions of the detection kit, and the absorbance value at a wavelength of 540 nm was measured using an ELISA reader.

[0064] (5) Milk protein content detection

[0065] The cell treatment method is the same as (3). The cell supernatant was collected and the α-lactalbumin and β-casein content was detected by enzyme-linked immunosorbent assay (ELISA), specifically according to the instructions of the detection kit, and the absorbance value at a wavelength of 450 nm was measured using an ELISA reader.

[0066] (6) Hormone receptor content detection

[0067] The cell treatment method is the same as (3). The cell supernatant is collected and the progesterone receptor and prolactin receptor content are detected by enzyme-linked immunosorbent assay (ELISA), and the specific operation is carried out according to the instructions of the detection kit, and the absorbance value at a wavelength of 450nm is measured using an enzyme marker.

[0068] 2. Test results

[0069] (1) Figure 1 The three functional substances in the present invention have an effect on the viability of canine mammary epithelial cells. The cell viability was determined by the CCK-8 method, and the cell survival rate of canine mammary cells after 1-320μg / mL perilla seed extract was 110%-160%; the cell survival rate of canine mammary cells after 1-320μg / mL alfalfa flavonoids was 90%-110%; the cell survival rate of canine mammary cells after 1-320μg / mL eucommia chlorogenic acid was 100%-140%. Therefore, 20μg / mL perilla seed extract, 20μg / mL eucommia chlorogenic acid and 20μg / mL alfalfa flavonoids were selected for subsequent experiments.

[0070] (2) Figure 2This is the effect of the combination of the three functional substances in the present invention on the concentration of lactose and lactase. The results showed that after the perilla seed extract, eucommia chlorogenic acid and alfalfa flavonoids acted on canine mammary epithelial cells for 24 hours, the lactose content increased, and the combination ratio (1-10): 1: (1-20) treatment was better than the single treatment group; the lactase content increased, and the combination ratio 20: 1: 1 and ratio 1: 1: (10-20) treatment was better than the single treatment group. This shows that the combination of the three substances has a synergistic effect.

[0071] (3) Figure 3 This is the effect of the combination of three functional substances in the present invention on the concentration of triglycerides and fatty acid synthase. The results showed that after perilla seed extract, eucommia chlorogenic acid and alfalfa flavonoids acted on canine mammary epithelial cells for 24 hours, the intracellular triglyceride content increased. When perilla seed: eucommia chlorogenic acid: alfalfa flavonoids were combined in a ratio of 20:1:1, the effect was more significant; eucommia chlorogenic acid can increase the concentration of fatty acid synthase, and when the ratio of the three substances was 1:20:1 and (10-20):1:1, the effect was better than that of single treatment. This shows that the three substances promote the production of milk fat in canine mammary cells.

[0072] (4) Figure 4 The results show that the combination of three functional substances in the present invention affects the concentration of α-lactalbumin (A) and β-casein (B). The results show that the 24h treatment of canine mammary epithelial cells with perilla seed extract, eucommia chlorogenic acid and alfalfa flavonoids increased the α-lactalbumin content, and the results of the combination treatment and the single treatment were similar; the β-casein content increased, and the combination ratio of (1-20):1:1 was better than the single treatment group.

[0073] (5) Figure 5 The effect of the combination of three functional substances in the present invention on the concentration of progesterone receptor and prolactin receptor. The results showed that after 24 hours of action of perilla seed extract, eucommia chlorogenic acid and alfalfa flavonoids on canine mammary epithelial cells, the progesterone receptor content increased, and the combination ratio of 1:20:1 and 20:1:1 was better than the single treatment group; the prolactin receptor content increased, and the combination treatment was better than the single treatment group, especially the ratio of 1:(1-10):(1-20). 2. Metabolomics analysis of the combination on canine mammary epithelial cells

[0074] 1. Test methods

[0075] In order to gain a deeper understanding of the metabolic differences of canine mammary epithelial cells by perilla seed extract, alfalfa flavonoids and eucommia chlorogenic acid, non-targeted metabolomics analysis was performed using high performance liquid chromatography tandem mass spectrometry (LC-MS). Untreated cells were used as the control group (BC), and cells treated with perilla seed extract (PSE), alfalfa flavonoids (AFF) and eucommia chlorogenic acid (CGA) were used as experimental groups. An equal volume of samples was taken from each experimental sample and mixed as the quality control group (QC). 53% methanol aqueous solution was used instead of the experimental sample as the blank control. The cell samples were placed in EP tubes, and 300 μL of 80% methanol aqueous solution was added; they were quickly frozen in liquid nitrogen for 5 minutes; they were vortexed for 30 seconds after thawing on ice, and ultrasonicated for 6 minutes; they were centrifuged at 5000 rpm and 4℃ for 1 minute, and the supernatant was taken into a new centrifuge tube and freeze-dried into dry powder; according to the volume of the sample, the corresponding 10% methanol solution was added to dissolve, and the sample was injected into LC-MS for analysis.

[0076] The samples were chromatographed on a Vanquish UHPLC instrument (Thermo Fisher) and a Q Exactive TM HF mass spectrometer (Thermo Fisher) was used for separation and analysis, and the scan range was selected as m / z 100-1500; the ESI source settings were as follows: Spray Voltage: 3.5 kV; Sheath gas flow rate: 35 psi; Aux Gas flow rate: 10 L / min; Capillary Temp: 320 °C; S-lens RF level: 60; Aux gas heater temp: 350 °C; Polarity: positive, negative; MS / MS secondary scans were data-dependent scans. The raw data were filtered by retention time and mass-to-charge ratio and normalized to the initial QC sample to obtain peak area accuracy. Peak extraction used parameters such as 5 ppm mass tolerance and 30% signal intensity deviation. Metabolites were identified by comparing molecular and fragment ion data with mzCloud, mzVault, and Masslist databases. Background ions were removed using blank samples. The quantitative results were normalized according to the ratio of the metabolite concentration in the sample to the metabolite concentration in the QC sample. Compounds with a coefficient of variation (CV) of more than 30% in the QC sample were excluded to finally complete the identification and relative quantification of the metabolites.

[0077] 2. Test results

[0078] Figure 6 It is a volcano plot of the differential metabolites between the groups in the present invention. Figure 7 It is a cluster heat map of differential metabolites between the groups in the present invention. The results showed that compared with the blank control group, 6 metabolites were upregulated and 25 metabolites were downregulated in the perilla seed extract treatment group; 9 metabolites were upregulated and 4 metabolites were downregulated in the alfalfa flavonoids treatment group; 5 metabolites were upregulated and 27 metabolites were downregulated in the eucommia chlorogenic acid treatment group. Among them, the contents of 4-guanidinobutyric acid, 2-isopropylmalic acid, and D-(-)-quinic acid related to amino acid synthesis and metabolism increased. After the cells were treated with perilla seed extract, alfalfa flavonoids and eucommia chlorogenic acid, the metabolite changes were opposite to those in the untreated state, with significant differences. The differential metabolites were annotated in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, the metabolic pathways involved were found, and the number of differential metabolites annotated in a certain pathway was enriched. The results showed that the biosynthesis and metabolic pathways of amino acids, glycerophospholipid metabolism, fatty acid biosynthesis and other pathways were significantly enriched. Therefore, perilla seed extract, alfalfa flavonoids and eucommia chlorogenic acid promoted canine mammary cell lactation by participating in these pathways.

[0079] According to the results of metabolomics analysis of canine mammary cells, some other substances should also be supplemented, such as butyric acid (regulates intestinal health, provides energy and nutrition, and inhibits inflammation), malic acid (promotes amino acid metabolism, which is beneficial to the health of the body), disodium 5'-guanylate (improves visual, olfactory, and taste conduction), benzoic acid and tartaric acid (have antibacterial and antioxidant effects), palm fatty acid powder (maintains cell membrane stability, promotes fatty acid synthesis, and provides energy for cells), nicotinamide (antioxidant effect, enhances the energy metabolism capacity of cells), cystine (antioxidant effect, protects cell membranes and DNA, and improves immunity), leucine (maintains the normal function of the nervous system and is also an important component of protein synthesis), tyrosine (is a precursor of adrenaline, dopamine, and norepinephrine, participates in nerve conduction and regulates emotions), lecithin (regulates cholesterol metabolism and balance, promotes cell metabolism and repair, and maintains nervous system function), linolenic acid and linoleic acid (anti-inflammatory and antioxidant, are important components of cell membranes, and are essential for maintaining normal cell function).

[0080] Table 1 Differential metabolites and metabolic pathways in canine mammary epithelial cells treated with perilla seed extract

[0081]

[0082]

[0083] Table 2 Differential metabolites and metabolic pathways in canine mammary epithelial cells treated with alfalfa flavonoids

[0084]

[0085]

[0086] Table 3 Differential metabolites and metabolic pathways in canine mammary epithelial cells treated with eucommia chlorogenic acid

[0087]

[0088]

[0089] Example 1

[0090] This embodiment provides a method for preparing a health-care agent for promoting lactation function of pets, which is carried out according to the following steps:

[0091] Step (1): weigh 3 parts of perilla seed extract, 3 parts of alfalfa flavonoids, and 3 parts of eucommia chlorogenic acid;

[0092] Step (2): Mix the Chinese herbal medicine weighed in step (1) with the additives, and add water until the mixed raw materials are submerged;

[0093] Step (3): After stirring evenly, granulation and air drying are performed to obtain a granular health-care agent.

[0094] The additives in step (2) are 2 parts of butyric acid, 2 parts of malic acid, 1 part of disodium 5'-guanylate, 1 part of benzoic acid, 2 parts of tartaric acid, 10 parts of palm fatty acid powder, 5 parts of niacinamide, 0.2 parts of cystine, 0.2 parts of leucine, 0.2 parts of tyrosine, 2 parts of lecithin, 6 parts of linolenic acid, and 5 parts of linoleic acid.

[0095] Example 2

[0096] This embodiment provides a method for preparing a health-care agent for promoting lactation function of pets, which is carried out according to the following steps:

[0097] Step (1): weigh 60 parts of perilla seed extract, 60 parts of alfalfa flavonoids, and 60 parts of eucommia chlorogenic acid;

[0098] Step (2): Mix the Chinese herbal medicine weighed in step (1) with the additives, and add water until the mixed raw materials are submerged;

[0099] Step (3): After stirring evenly, granulation and air drying are performed to obtain a granular health-care agent.

[0100] The additives in step (2) are 1 part of butyric acid, 1 part of malic acid, 0.5 part of disodium 5'-guanylate, 0.5 part of benzoic acid, 1 part of tartaric acid, 5 parts of palm fatty acid powder, 3 parts of niacinamide, 0.1 part of cystine, 0.1 part of leucine, 0.1 part of tyrosine, 1 part of lecithin, 4 parts of linolenic acid, and 2 parts of linoleic acid.

[0101] Example 3

[0102] This embodiment provides a method for preparing a health-care agent for promoting lactation function of pets, which is carried out according to the following steps:

[0103] Step (1): weigh 60 parts of perilla seed extract, 3 parts of alfalfa flavonoids, and 3 parts of eucommia chlorogenic acid;

[0104] Step (2): Mix the Chinese herbal medicine weighed in step (1) with the additives, and add water until the mixed raw materials are submerged;

[0105] Step (3): After stirring evenly, granulation and air drying are performed to obtain a granular health-care agent.

[0106] The additives in step (2) are 2 parts of butyric acid, 2 parts of malic acid, 1 part of disodium 5'-guanylate, 1 part of benzoic acid, 2 parts of tartaric acid, 10 parts of palm fatty acid powder, 5 parts of niacinamide, 0.2 parts of cystine, 0.2 parts of leucine, 0.2 parts of tyrosine, 2 parts of lecithin, 6 parts of linolenic acid, and 5 parts of linoleic acid.

[0107] Example 4

[0108] This embodiment provides a method for preparing a health-care agent for promoting lactation function of pets, which is carried out according to the following steps:

[0109] Step (1): weigh 3 parts of perilla seed extract, 3 parts of alfalfa flavonoids, and 60 parts of eucommia chlorogenic acid;

[0110] Step (2): Mix the Chinese herbal medicine weighed in step (1) with the additives, and add water until the mixed raw materials are submerged;

[0111] Step (3): After stirring evenly, granulation and air drying are performed to obtain a granular health-care agent.

[0112] The additives in step (2) are 2 parts of butyric acid, 2 parts of malic acid, 1 part of disodium 5'-guanylate, 1 part of benzoic acid, 2 parts of tartaric acid, 10 parts of palm fatty acid powder, 5 parts of niacinamide, 0.2 parts of cystine, 0.2 parts of leucine, 0.2 parts of tyrosine, 2 parts of lecithin, 6 parts of linolenic acid, and 5 parts of linoleic acid.

[0113] Comparative Example 1

[0114] The difference from Example 1 is that no perilla seed extract, alfalfa flavonoids and eucommia chlorogenic acid are added.

[0115] Comparative Example 2

[0116] The difference from Example 1 is that butyric acid, malic acid, disodium 5'-guanylate, benzoic acid, tartaric acid, palm fatty acid powder, nicotinamide, cystine, leucine, tyrosine, lecithin, linolenic acid and linoleic acid are not added.

[0117] Feeding effect experiment:

[0118] 21 healthy dogs with similar milk production and no mastitis were randomly divided into 7 groups, and the compositions of the above embodiments and comparative examples were added to the dog diets for 7 consecutive days, and the control group was only fed with ordinary dog ​​food. The milk production of the dogs was tested every day.

[0119] The milk production of each group of dogs is shown in Table 4 below. After feeding Examples 1 to 4, the milk production of each group of dogs began to increase on the 3rd day, which was 17.6% to 31.2% higher than that of the control group. After feeding Comparative Examples 1 to 2, the milk production of each group of dogs began to increase on the 4th day, which was 10.2% to 18.8% higher than that of the control group. Although Comparative Examples 1 to 2 also increased the milk production of dogs, the effect was not as significant as that of Examples 1 to 4. This shows that the perilla seed extract, alfalfa flavonoids and eucommia chlorogenic acid in the present invention can quickly act on canine mammary cells, increase the concentration of lactose, milk protein and milk fat, promote lactation, and solve the problem of insufficient milk production in female dogs, that is, the various substances in the formula of the present invention can better increase the milk production of dogs through mutual synergistic effects.

[0120] Table 4 Milk production of dogs fed with the compositions of the examples and comparative examples

[0121]

Claims

1. A composition for promoting lactation of pets, characterized in that The composition comprises perilla seed extract, alfalfa flavonoids and eucommia chlorogenic acid, and the weight proportions thereof are as follows: The perilla seed extract is 3 to 60 parts, the alfalfa flavonoids is 3 to 60 parts, and the eucommia chlorogenic acid is 3 to 60 parts.

2. A composition for promoting lactation of pets according to claim 1, characterized in that The composition comprises perilla seed extract, alfalfa flavonoids and eucommia chlorogenic acid, and the weight proportions thereof are as follows: The perilla seed extract is 60 parts, the alfalfa flavonoids are 3 parts, and the eucommia chlorogenic acid is 3 parts.

3. A health care agent for promoting lactation function of pets, characterized in that It is composed of the following raw materials in parts by weight: 3-60 parts of perilla seed extract, 3-60 parts of alfalfa flavonoids, 3-60 parts of eucommia chlorogenic acid, 1-2 parts of butyric acid, 1-2 parts of malic acid, 0.5-1 parts of disodium 5'-guanylate, 0.5-1 parts of benzoic acid, 1-2 parts of tartaric acid, 5-10 parts of palm fatty acid powder, 3-5 parts of niacinamide, 0.1-0.2 parts of cystine, 0.1-0.2 parts of leucine, 0.1-0.2 parts of tyrosine, 1-2 parts of lecithin, 4-6 parts of linolenic acid, and 2-5 parts of linoleic acid.

4. The health care agent for promoting lactation function of pets according to claim 3, characterized in that It is composed of the following raw materials in parts by weight: 60 parts of perilla seed extract, 3 parts of alfalfa flavonoids, 3 parts of eucommia chlorogenic acid, 2 parts of butyric acid, 2 parts of malic acid, 1 part of disodium 5'-guanylate, 1 part of benzoic acid, 2 parts of tartaric acid, 10 parts of palm fatty acid powder, 5 parts of niacinamide, 0.2 parts of cystine, 0.2 parts of leucine, 0.2 parts of tyrosine, 2 parts of lecithin, 6 parts of linolenic acid, and 5 parts of linoleic acid.

5. The composition for promoting lactation of pets according to claim 1 or the health care agent for promoting lactation of pets according to claim 3, characterized in that The perilla seed extract is prepared according to the following steps: Step (1): weigh perilla seeds, crush them, add 60% ethanol, extract them under reflux at 55° C. for 2 times, each time for 1 hour, filter, and obtain a perilla seed extract; wherein the mass to volume ratio of perilla seeds to 60% ethanol is 1:10 (g / mL). Step (2): The perilla seed extract is concentrated to dryness under reduced pressure using a vacuum concentrator pump to obtain a perilla seed extract.

6. The composition for promoting lactation of pets according to claim 1 or the health care agent for promoting lactation of pets according to claim 3, characterized in that The alfalfa flavonoids are prepared according to the following steps: Step (1): Wash and dry fresh alfalfa, then grind it into powder, add an appropriate amount of sodium hydroxide solution, reflux extract in a constant temperature water bath at 50° C. for 2 hours, filter, and obtain an extract; The mass-to-volume ratio of dry alfalfa to sodium hydroxide solution is 1:10 (g / mL); Step (2): adding 60% ethanol solution to the extract, extracting at 80° C. for 1 hour, and centrifuging and drying to obtain alfalfa flavonoids; wherein the volume ratio of the extract to 60% ethanol is 1:10 (mL / mL).

7. The composition for promoting lactation of pets according to claim 1 or the health care agent for promoting lactation of pets according to claim 3, characterized in that The Eucommia ulmoides chlorogenic acid is prepared according to the following steps: Step (1): crushing the Eucommia leaves, adding 70% ethanol, extracting at 60° C. for 3 times, each time for 1 hour, filtering to obtain an extract; wherein the mass to volume ratio of the Eucommia leaves to the 70% ethanol is 1:30 (g / mL); Step (2): The extract is concentrated to dryness using a vacuum concentration and pressure reduction pump to obtain Eucommia ulmoides chlorogenic acid.

8. The method for preparing a health-care agent for promoting lactation function of pets according to claim 3, characterized in that Follow the steps below: Step (1): weighing 3-60 parts of perilla seed extract, 3-60 parts of alfalfa flavonoids, and 3-60 parts of eucommia chlorogenic acid; Step (2): Mix the Chinese herbal medicine weighed in step (1) with the additives, and add water until the mixed raw materials are submerged; Step (3): After stirring evenly, granulation and air drying are performed to obtain a granular health-care agent.

9. The method for preparing a health-care agent for promoting lactation function of pets according to claim 8, characterized in that The additive in step (2) is one or more of butyric acid, malic acid, disodium 5'-guanylate, benzoic acid, tartaric acid, palm fatty acid powder, nicotinamide, cystine, leucine, tyrosine, lecithin, linolenic acid and linoleic acid; The additives in step (2) are 1-2 parts of butyric acid, 1-2 parts of malic acid, 0.5-1 parts of disodium 5'-guanylate, 0.5-1 parts of benzoic acid, 1-2 parts of tartaric acid, 5-10 parts of palm fatty acid powder, 3-5 parts of niacinamide, 0.1-0.2 parts of cystine, 0.1-0.2 parts of leucine, 0.1-0.2 parts of tyrosine, 1-2 parts of lecithin, 4-6 parts of linolenic acid, and 2-5 parts of linoleic acid.

10. Use of the composition for promoting lactation function of pets according to claims 1-2 or the health care agent for promoting lactation function of pets according to claims 3-4 for promoting proliferation of canine mammary epithelial cells, or for increasing the intracellular lactose, milk protein and milk fat content.

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