Preparation method and application of 7-hydroxyisoflavone acetylferulate

Abstract

The invention discloses a preparation method and application of 7-hydroxyisoflavone acetylferulate, relating to the technical field of feed additives. The preparation method comprises the following steps: adding acetylferulic acid, 7-hydroxyisoflavone, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N,N-dimethyl-4-pyridinamine to a reactor; using dichloromethane as a solvent; stirring at room temperature for 5 hours; adding water after the reaction is complete; and then extracting with dichloromethane; and concentrating and crystallizing the product to obtain a target product. The product prepared by this method has good fat solubility and stability, and can be added as an additive to livestock, poultry, and aquatic animal feed to significantly improve the growth performance, antioxidant capacity, immunity, and intestinal digestion capacity of the animal.

Description

Technical Field

[0001] The present invention relates to the technical field of feed additives, and in particular to a preparation method and application of a novel feed additive. The feed additive is mainly suitable for livestock, poultry and aquatic animals. Background Art

[0002] The rise of antibiotics in feed has ushered in a new era in the development of animal husbandry. However, the overuse and excessive use of antibiotics severely hinders the sustainable and green development of my country's animal husbandry industry. The misuse of antibiotics will lead to widespread outbreaks of drug-resistant bacteria and the emergence of superbugs resistant to multiple antibiotics. Currently, the country has introduced a series of policies to gradually phase out antibiotics from the feed additive market, and antibiotics are no longer suitable for the new animal husbandry system. A green and environmentally friendly antibiotic-free animal husbandry system will gradually become the mainstream trend in the animal husbandry industry. Therefore, the development of residual-free and drug-resistant feed additives has become a top priority for the animal husbandry industry in this new era. A new generation of antibiotic alternatives will bring new benefits to modern animal husbandry systems.

[0003] Ferulic acid is a phenolic acid extracted from common plant species. It is one of the main active monomeric components of various water-soluble extracts of traditional Chinese medicines (such as Ferula, Angelica sinensis, Cimicifuga heracleifolia, and Taraxacum mongolicum). It exhibits potent antioxidant, antibacterial, anti-inflammatory, antithrombotic, anticancer, analgesic, and immune-enhancing properties. However, ferulic acid has poor lipid solubility and is difficult to penetrate the lipid bilayer of biological membranes, resulting in low bioavailability. Therefore, structural modification of ferulic acid to produce ferulic acid derivatives is necessary to enhance its therapeutic effects. 7-Hydroxyisoflavone is a flavonoid compound that is readily soluble in weakly polar solutions such as alcohols and their aqueous solutions, such as methanol and ethanol, but poorly soluble in water. It has physiological functions such as promoting animal growth and reproduction, enhancing antioxidant capacity, immunity, and intestinal digestion and absorption. Summary of the Invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides a method for preparing a novel feed additive which has good fat solubility, can effectively improve the growth performance, antioxidant capacity, immunity and intestinal digestion capacity of animals, and has a simple method and high yield.

[0005] In order to solve the above technical problems, a method for preparing 7-hydroxyisoflavone acetylferulate of the present invention comprises the steps of:

[0006] (1) Add acetylferulic acid, 7-hydroxyisoflavone, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N, N-dimethyl-4-pyridinamine into a reactor, use dichloromethane as solvent, stir at room temperature for 5 hours, add a large amount of water, extract with dichloromethane, concentrate, and crystallize to obtain the target product.

[0007] The structure of the 7-hydroxyisoflavone acetylferulate of the present invention is shown in the following formula (I):

[0008]

[0009] (I)

[0010] The mass ratio of acetylferulic acid, 7-hydroxyisoflavone, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N, N-dimethyl-4-pyridinamine in the above step (1) of the present invention is 1.0:1.2:(1.0-2.0):(0.06-0.6).

[0011] Preferably, the mass ratio of acetylferulic acid, 7-hydroxyisoflavone, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N, N-dimethyl-4-pyridinamine in step (1) is 1.0:1.2:1.4:0.06.

[0012] Preferably, a large amount of water is added in step (1), and the volume of water is 300 to 1000 mL.

[0013] The present invention further provides the use of the 7-hydroxyisoflavone acetylferulate in preparing feed for at least one of livestock, poultry and aquatic animals.

[0014] In the above application, the aquatic animal is at least one of tilapia, grass carp, largemouth bass and crayfish;

[0015] The usage amount of the 7-hydroxyisoflavone acetylferulate of the present invention is 100-1000 mg / kg feed (preferred range).

[0016] Furthermore, the dosage of the 7-hydroxyisoflavone acetylferulate of the present invention is 100-500 mg / kg feed (optimal range).

[0017] Furthermore, the dosage of the 7-hydroxyisoflavone acetylferulate of the present invention is 200 mg / kg feed (optimal specific point).

[0018] In the present invention, the 7-hydroxyisoflavone acetylferulate is added to the feed at a ratio of 200 mg / kg of feed, and the premix must be premixed into the base material by stepwise expansion.

[0019] Compared with the prior art, the present invention has the following characteristics:

[0020] 1) The method of the present invention is simple to synthesize and has a high yield of 80-88%.

[0021] 2) The reagents used in the method of the present invention are low in toxicity, have a fast reaction speed, and have low requirements for reaction equipment.

[0022] 3) The 7-hydroxyisoflavone acetylferulate of the present invention can be used in livestock, poultry and aquatic animals, especially aquatic animals such as tilapia, grass carp, largemouth bass and Procambarus clarkii.

[0023] 4) The 7-hydroxyisoflavone acetylferulate of the present invention has good fat solubility and stability, and is added to feed to effectively improve the growth performance, antioxidant capacity, immunity and intestinal digestion capacity of aquatic animals.

[0024] 5) When the dosage of 7-hydroxyisoflavone acetylferulate of the present invention is less than 100 mg / kg, the health improvement effect on fish and shrimp is not obvious; when the dosage of 7-hydroxyisoflavone acetylferulate is higher than 1000 mg / kg, the cost of feed is significantly increased; the optimal addition amount of 7-hydroxyisoflavone acetylferulate is 200 mg / kg, which does not significantly increase the cost while effectively improving the growth performance, antioxidant capacity, immunity and intestinal digestion capacity of aquatic animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a molecular structure diagram of the product of the present invention;

[0026] Figure 2 This is the infrared spectrum of the product prepared according to the embodiment of the present invention. Implementation Method

[0027] The present invention is further described in detail below through examples, but the present invention is not limited to the following examples. Any reasonable changes based on the technical solutions of the present invention are considered to fall within the scope of protection of the present invention. Example 1

[0028] 116.8 g of 7-hydroxyisoflavone, 116.8 g of acetylferulic acid, 116.8 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 7.0 g of N, N-dimethyl-4-pyridinamine were added to a reactor, and then 1000 mL of dichloromethane was added. The mixture was stirred at room temperature for 5 hours, 300 mL of water was added, and the mixture was extracted with dichloromethane, dried, recrystallized with ethanol, and dried to obtain 200.6 g of the target product. After nuclear magnetic resonance detection, the specific test results were as follows: 1H NMR (CHCl3, TMS, 400 MHz): δ=8.30-8.28 (d, 1H), 7.95 (s, 1H), 7.79 (d, 1H), 7.51 (d, 1H), 7.38-7.34 (m,2H), 7.33-7.30 (m, 4H), 7.15-7.13 (m, 2H), 7.10-7.03 (m, 1H), 6.54-6.50 (d,1H), 3.82 (s, 3H), 2.27 (s, 3H); 13 CNMR (CDCl3, 100 MHz) δ 175.27, 168.29,164.02, 156.58, 154.59, 153.21, 151.21, 146.94, 142.05, 132.95, 131.40,127.84, 125.77, 123.73, 121.39, 119.49, 116.61,116.25, 111.71,110.84, 102.90,55.94, 20.91. After infrared detection, the specific test results are: IR (KBr, cm -1 ): 3677 cm -1 ʋ(OH), 1772cm -1 ʋ(COOC-), 1747 cm -1 ʋ(COOH), 1652 cm -1 ʋ(C=O), 1130 cm -1 ʋ(COC), the infrared spectrum of the product is as shown in the attached Figure 2 According to the results of nuclear magnetic resonance detection and infrared spectrum, the target product was determined to be 7-hydroxyisoflavone acetylferulate. Example 2

[0029] 116.8 g of 7-hydroxyisoflavone, 116.8 g of acetylferulic acid, 233.6 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 70.1 g of N, N-dimethyl-4-pyridinamine were added to a reactor, and 600 mL of dichloromethane was added. The mixture was stirred at room temperature for 5 hours, and 1000 mL of water was added. The mixture was extracted with dichloromethane, dried, recrystallized with ethanol, and dried to obtain 192.6 g of the target product. After nuclear magnetic resonance detection, the specific test results were as follows: 1H NMR (CHCl3, TMS, 400 MHz): δ=8.30-8.28 (d, 1H), 7.95 (s, 1H), 7.79 (d, 1H), 7.51 (d, 1H), 7.38-7.34 (m,2H), 7.33-7.30 (m, 4H), 7.15-7.13 (m, 2H), 7.10-7.03 (m, 1H), 6.54-6.50 (d,1H), 3.82 (s, 3H), 2.27 (s, 3H); 13 CNMR (CDCl3, 100 MHz) δ 175.27, 168.29,164.02, 156.58, 154.59, 153.21, 151.21, 146.94, 142.05, 132.95, 131.40,127.84, 125.77, 123.73, 121.39, 119.49, 116.61,116.25, 111.71,110.84, 102.90,55.94, 20.91. Example 3

[0030] 116.8 g of 7-hydroxyisoflavone, 116.8 g of acetylferulic acid, 175.2 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 35.4 g of N, N-dimethyl-4-pyridinamine were added to a reactor, followed by 300 mL of dichloromethane. The mixture was stirred at room temperature for 5 hours, 1000 mL of water was added, and the mixture was extracted with dichloromethane, dried, recrystallized from ethanol, and dried to obtain 187.6 g of the target product. Example 4

[0031] 120 g of 7-hydroxyisoflavone, 120 g of acetylferulic acid, 204 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 66 g of N, N-dimethyl-4-pyridinamine were added to a reactor, followed by 800 mL of dichloromethane. The mixture was stirred at room temperature for 5 hours, 600 mL of water was added, and the mixture was extracted with dichloromethane, dried, recrystallized from ethanol, and dried to obtain 203.1 g of the target product. Example 5

[0032] 100 g of 7-hydroxyisoflavone, 100 g of acetylferulic acid, 165 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 45 g of N, N-dimethyl-4-pyridinamine were added to a reactor, followed by 800 mL of dichloromethane. The mixture was stirred at room temperature for 5 hours, 500 mL of water was added, and the mixture was extracted with dichloromethane, dried, recrystallized from ethanol, and dried to obtain 178.6 g of the desired product.

[0033] Application Example 1

[0034] The application effect of 7-hydroxyisoflavone acetylferulate in tilapia feed:

[0035] 7-Hydroxyisoflavone acetylferulate (200 mg / kg) was added to the basal diet. Two treatment groups, each with four replicates, were stocked with 30 juvenile tilapia. After eight weeks of culture in an indoor recirculating aquaculture system, growth indicators, serum biomarkers, and intestinal digestibility were measured.

[0036] Table 1 Effects of additive 7-hydroxyisoflavone acetylferulate on growth performance of tilapia

[0037] Experimental group Initial body mass (g) Final body mass (g) Weight gain rate (%) Feed coefficient control group 12.11 112.72 830.80 1.24 Additive Group 12.14 128.24 956.34 1.12

[0038] Table 2 Effects of the additive 7-hydroxyisoflavone acetylferulate on serum parameters and intestinal protease activity of tilapia

[0039] Experimental group Albumin / (g / L) Lysozyme / (U / mL) Catalase / (U / mL) Superoxide dismutase / (U / mL) Protease / (U / g) control group 10.25 15.81 21.43 53.76 521.41 Additive Group 14.83 25.62 25.95 70.43 588.96

[0040] The above results show that 7-hydroxyisoflavone acetylferulate significantly improves the growth performance of tilapia fry, and effectively enhances its antioxidant capacity, immunity and intestinal digestion ability.

[0041] Application Example 2

[0042] Effect of application of 7-hydroxyisoflavone acetylferulate in grass carp feed:

[0043] 7-Hydroxyisoflavone acetylferulate (200 mg / kg) was added to the basal diet. Thirty grass carp were stocked in each of two treatment groups, each with four replicates. After eight weeks of culture in an indoor recirculating aquaculture system, growth indicators, serum biomarkers, and intestinal digestibility were measured.

[0044] Table 3 Effects of the additive 7-hydroxyisoflavone acetylferulate on the growth performance of grass carp

[0045] Experimental group Initial body mass (g) Final body mass (g) Weight gain rate (%) Feed coefficient control group 12.34 90.66 634.68 1.25 Additive Group 12.25 101.82 731.18 1.13

[0046] Table 4 Effects of the additive 7-hydroxyisoflavone acetylferulate on serum parameters and intestinal protease activity of grass carp

[0047] Experimental group Albumin / (g / L) Lysozyme / (U / mL) Catalase / (U / mL) Superoxide dismutase / (U / mL) Protease / (U / g) control group 110.32 315.66 11.40 43.77 121.36 Additive Group 140.22 355.21 15.55 54.43 144.87

[0048] The above results show that the additive 7-hydroxyisoflavone acetylferulate significantly improved the growth performance of grass carp, and effectively enhanced its antioxidant capacity, immunity and intestinal digestion ability.

[0049] Application Example 3

[0050] Effect of application of 7-hydroxyisoflavone acetylferulate in largemouth bass feed:

[0051] 7-Hydroxyisoflavone acetylferulate was added to the basal diet. Two treatment groups, each with four replicates, were stocked with 30 largemouth bass in each replicate. After eight weeks of culture in an indoor recirculating aquaculture system, growth indicators, serum biomarkers, and intestinal digestibility were measured.

[0052] Table 5 Effects of the additive 7-hydroxyisoflavone acetylferulate on the growth performance of largemouth bass

[0053] Experimental group Initial body mass (g) Final body mass (g) Weight gain rate (%) Feed coefficient control group 6.55 38.16 482.60 0.99 Additive Group 6.60 41.64 530.91 0.89

[0054] Table 6 Effects of the additive 7-hydroxyisoflavone acetylferulate on serum parameters and intestinal protease activities of largemouth bass

[0055] Experimental group Albumin / (g / L) Lysozyme / (U / mL) Catalase / (U / mL) Superoxide dismutase / (U / mL) Protease / (U / g) control group 6.32 912.54 31.22 145.36 325.85 Additive Group 7.44 1154.88 37.59 154.24 384.22

[0056] The above results show that 7-hydroxyisoflavone acetylferulate significantly improved the growth performance of largemouth bass, and effectively enhanced its antioxidant capacity, immunity and intestinal digestion ability.

[0057] Application Example 4

[0058] Effect of application of 7-hydroxyisoflavone acetylferulate in crayfish feed:

[0059] 7-Hydroxyisoflavone acetylferulate was added to the basal diet. Two treatment groups, each with four replicates, were stocked with 15 Procambarus clarkii in each replicate. After eight weeks of culture in cages, growth indicators, serum parameters, and intestinal digestion capacity were measured.

[0060] Table 7 Effects of 7-hydroxyisoflavone acetylferulate on growth performance of Procambarus clarkii

[0061] Experimental group Initial body mass (g) Final body mass (g) Weight gain rate (%) Feed coefficient control group 9.22 18.21 97.51 3.10 Additive Group 9.18 20.02 118.08 2.82

[0062] Table 8 Effects of 7-hydroxyisoflavone acetylferulate on serum parameters and intestinal protease activity of Procambarus clarkii

[0063] Experimental group Albumin / (g / L) Lysozyme / (U / mL) Catalase / (U / mL) Superoxide dismutase / (U / mL) Protease / (U / g) control group 36.66 2.55 10.35 35.22 60.44 Additive Group 47.89 3.89 12.67 40.64 65.81

[0064] The above results show that 7-hydroxyisoflavone acetylferulate significantly improved the growth performance of Procambarus clarkii, while effectively enhancing its antioxidant capacity, immunity and intestinal digestion ability.

Claims

1. An application of 7-hydroxyisoflavone acetylferulate, characterized in that: 7-Hydroxyisoflavone acetylferulate is used as an additive in livestock, poultry and aquatic animal feed. 7-Hydroxyisoflavone acetylferulate has the following structural formula: 。 2. The use of 7-hydroxyisoflavone acetylferulate according to claim 1, characterized in that: The aquatic animals are one of the following: tilapia, grass carp, largemouth bass and Procambarus clarkii.

3. The use of 7-hydroxyisoflavone acetylferulate according to claim 1, characterized in that: The dosage of 7-hydroxyisoflavone acetylferulate is 100-1000 mg / kg feed.

4. The use of 7-hydroxyisoflavone acetylferulate according to claim 3, characterized in that: The dosage of 7-hydroxyisoflavone acetylferulate is 200-500 mg / kg feed.

5. The use of 7-hydroxyisoflavone acetylferulate according to claim 3, characterized in that: The dosage of 7-hydroxyisoflavone acetylferulate was 200 mg / kg feed.

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