Application of compound and composition thereof in preventing and treating poultry from laying soft-shell eggs and broken-shell eggs

Abstract

The invention discloses an application of a compound and a composition thereof in preventing and treating poultry from laying soft-shell eggs and broken-shell eggs. The chemical structural formula of the compound is # imgabs0, and the compound is prepared by extracting abelmoschus manihot flowers and separating and purifying the abelmoschus manihot flowers. The compound can significantly reduce the rate of soft eggshells and broken eggshells. According to the composition of the compound and quercetin, the rate of soft eggshells and broken eggshells can be obviously reduced, meanwhile, the daily laying rate can be obviously increased, the feed-egg ratio can be reduced, the egg laying performance of poultry can be improved, and the economic benefits of poultry farmers can be increased.

Description

Technical Field

[0001] The present invention belongs to the field of veterinary drugs, and particularly relates to the use of a compound and its composition in preventing and treating soft-shell eggs and cracked eggs in poultry. Background Art

[0002] At present, the food safety problem in China is quite serious. Due to the abuse of veterinary drugs, drugs such as antibiotics and hormones accumulate in poultry, resulting in excessive drug residues in the eggs produced by poultry, seriously affecting the quality of egg products. In recent years, veterinary drugs that obtain natural, non-antibiotic hormones from traditional Chinese medicine and improve the egg-laying performance of poultry have become a research hotspot. However, compared with the huge number of traditional Chinese medicines, only a very small number of traditional Chinese medicines are used in veterinary drugs. It is of great significance to explore traditional Chinese medicines with the potential to become veterinary drugs.

[0003] After the peak egg-laying period, as the age of poultry increases, their physiological functions gradually decline. Especially in the late egg-laying period, the egg-laying rate decreases, the eggshell quality deteriorates, and the rates of soft-shell eggs and cracked eggs increase significantly. At the same time, the feed intake of poultry increases and the economic benefits decrease.

[0004] Flavonoids are polyphenolic substances with a C6-C3-C6 structure existing in plants, having various physiological functions and wide application values in the fields of medicine, food and health products. Flavonoid compounds improve the reproductive ability of animals by affecting hormones related to reproduction in livestock and poultry. In the prior art, there have been reports on adding flavonoid compounds such as soy isoflavone and quercetin, and polyphenolic compounds such as curcumin and gallnut tannin to feed to improve the production performance of poultry and egg quality.

[0005] Hibiseu manihot L., also known as Abelmoschus manihot Medic., golden hibiscus, is an annual herbaceous plant of the genus Abelmoschus in the family Malvaceae. Its flowers are rich in flavonoid compounds and are ideal parts for medicinal use. Summary of the Invention

[0006] Aiming at the deficiencies of the prior art, the object of the present invention is to prepare a compound and its composition, and develop the compound and its composition into a veterinary drug for preventing and treating soft-shell eggs and cracked eggs in poultry and improving the egg-laying performance of poultry.

[0007] The compound of the present invention is characterized in that its chemical structure is as follows:

[0008]

[0009] The preparation method of the compound of the present invention includes the following steps:

[0010] (1) Extracting the flowers of Hibiseu manihot L. with 70% ethanol, and concentrating the extract until there is no alcohol smell;

[0011] (2) The concentrated solution was successively extracted with petroleum ether, dichloromethane, and ethyl acetate. The extracts were concentrated under reduced pressure to obtain extracts, namely petroleum ether extract, dichloromethane extract, and ethyl acetate extract respectively.

[0012] (3) The ethyl acetate extract was further separated by silica gel column chromatography. It was eluted with a mixed solution of ethyl acetate and ethanol with a volume ratio of 25:1, and the eluate was discarded. Then it was eluted with a mixed solution of ethyl acetate and ethanol with a volume ratio of 15:1, and the eluate was collected and concentrated to an extract.

[0013] (4) The extract was dissolved in methanol and further purified by Sephadex LH-20 column chromatography. Using 75% ethanol solution as the mobile phase, the orange band was collected, the solvent was recovered, and this step was repeated until the purity detected by high performance liquid chromatography reached over 98%.

[0014] The compound of the present invention can significantly reduce the rates of soft eggshells and broken eggshells, and is used for preventing and treating soft-shelled eggs and broken eggs in poultry.

[0015] The composition of the present invention includes the compound of the present invention and quercetin. The proportion of the composition is 0.05 - 0.2 parts of the compound of the present invention and 1 part of quercetin, and the preferred proportion is 0.1 part of the compound of the present invention and 1 part of quercetin.

[0016] The composition of the present invention can improve the egg-laying performance of poultry while preventing and treating soft-shelled eggs and broken eggs in poultry. Description of the Drawings

[0017] Figure 1 For the nuclear magnetic resonance 1 1H-NMR spectrum

[0018] Figure 2 For the nuclear magnetic resonance 13 13C-NMR spectrum

[0019] Figure 3 For the HSQC spectrum of the nuclear magnetic resonance of the compound of the present invention

[0020] Figure 4 For the HMBC spectrum of the nuclear magnetic resonance of the compound of the present invention Detailed Embodiments

[0021] The present invention will be further described below in conjunction with embodiments, but it does not limit the implementation of the present invention.

[0022] Example 1

[0023] The effects of the compound of the present invention and its composition with quercetin on the egg-laying performance of Hy-Line brown laying hens in the late laying period

[0024] 1.1 Experimental animals: 500 55-week-old Hy-Line Brown laying hens were randomly divided into 10 groups, namely the control group, the low-dose compound group, the medium-dose compound group, the high-dose compound group, the low-dose quercetin group, the medium-dose quercetin group, the high-dose quercetin group, composition group 1 (0.05 parts of compound and 1 part of quercetin), composition group 2 (0.1 part of compound and 1 part of quercetin), and composition group 3 (0.2 part of compound and 1 part of quercetin), as shown in Table 1. Each group had 5 replicates, with 10 chickens in each replicate. The preliminary trial period was 3 weeks, during which the chickens were fed a basal diet, as shown in Table 2. The experimental period was 8 weeks in total.

[0025] Table 1 Experimental design

[0026]

[0027] Table 2 Composition and nutrient levels of the basal diet

[0028]

[0029] a The premix provided 9000 IU of VA, 2500 IU of VD, 20 IU of VE, 12 μg of VB12, and 2.4 mg of VK per kg of diet; trace elements included 100 mg of Mn, 60 mg of Zn, 25 mg of Fe, 10 mg of Cu, 0.35 mg of Se (N2SeO3·5H2O), and 0.5 mg of I (KI).

[0030] 1.2 Reagents and main instruments: The estradiol detection kit and progesterone detection kit were both purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd.

[0031] Analytical balance (Mettler), tabletop centrifuge (Shanghai Anting), constant temperature water bath, and Multiskan FC microplate reader (Sanofi).

[0032] 1.3 Determination methods

[0033] 1.3.1 The determination methods for each production performance index are as follows:

[0034] (1) Daily egg production rate = total number of eggs produced during the statistical period ÷ (number of feeding days × number of laying hens) × 100%

[0035] (2) Average egg weight = total egg weight ÷ total number of eggs produced

[0036] (3) Daily feed intake = feed consumption during the statistical period ÷ (number of feeding days × number of laying hens)

[0037] (4) Feed-to-egg ratio (feed conversion rate) = total feed consumption during the statistical period ÷ total egg weight.

[0038] (5) Soft and cracked eggshell rate = number of soft-shelled and cracked eggs ÷ total number of eggs produced × 100%

[0039] 1.3.2 Determination of endocrine hormone indicators

[0040] After the feeding trial, 1 laying hen was randomly selected from each replicate of each group, blood was collected from the anterior vena cava, serum was separated and stored in a -30°C refrigerator.

[0041] Serum estradiol and progesterone were determined by enzyme-linked immunosorbent assay.

[0042] 1.4 The experimental data was processed using SPSS 20.0 software, and all data statistical analysis was performed using represented, and one-way analysis of variance (One-Way-ANOVA) was used for comparison among multiple groups. P < 0.05 indicated statistical significance.

[0043] 1.5 Index detection and experimental results

[0044] 1.5.1 Production performance

[0045] Compared with the control group, adding the compound described in the present invention to the diet could reduce the rates of soft-shell eggs and cracked eggs. With the increase of the addition level, the rates of soft-shell eggs and cracked eggs decreased significantly, and the difference was statistically significant; adding medium and high doses of the compound to the diet could increase the egg production rate, and the difference was statistically significant; adding the compound to the diet had no significant effect on the average egg weight, daily feed intake, and feed-to-egg ratio.

[0046] Compared with the control group, adding quercetin to the diet could increase the daily egg production rate. With the increase of the addition level, the daily egg production rate increased significantly, and the difference was statistically significant; adding quercetin to the diet could reduce the feed-to-egg ratio, and the difference was statistically significant; adding quercetin to the diet had no significant effect on the average egg weight, daily feed intake, and the rates of soft-shell eggs and cracked eggs.

[0047] Compared with the control group, adding the composition of the compound described in the present invention and quercetin to the diet could increase the daily egg production rate. The optimal combination ratio was 0.1 part of the compound and 1 part of quercetin, and the difference was statistically significant; adding the composition of the compound described in the present invention and quercetin to the diet could reduce the rates of soft-shell eggs and cracked eggs. The optimal combination ratio was 0.1 part of the compound and 1 part of quercetin, and the difference was statistically significant; adding the composition of the compound described in the present invention and quercetin to the diet could reduce the feed-to-egg ratio, and the difference was statistically significant; adding the composition of the compound described in the present invention and quercetin to the diet had no significant effect on the average egg weight and daily feed intake.

[0048] See Table 3 for details.

[0049] Table 3 Effects of the compound described in the present invention, quercetin and their combination on the laying performance of Hy-Line brown laying hens in the late laying period

[0050]

[0051] Note: "*" indicates P < 0.05 compared with the control group, and "**" indicates P < 0.01 compared with the control group.

[0052] 1.5.2 Serum hormone determination

[0053] Compared with the control group, adding the compound described in the present invention to the diet can significantly increase the serum estradiol and progesterone levels of Hy-Line Brown laying hens in the late laying period, and the difference is statistically significant; adding quercetin to the diet can significantly increase the serum estradiol and progesterone levels of Hy-Line Brown laying hens in the late laying period, and the difference is statistically significant; when the combination ratio of each group is 0.1 part of the compound and 1 part of quercetin, the increase in estradiol and progesterone levels is the best, as shown in Table 4 for details.

[0054] Table 4 Effects of the compound, quercetin and their combination described in the present invention on the serum hormone levels of Hy-Line Brown laying hens in the late laying period

[0055]

[0056]

[0057] Note: "*" indicates P < 0.05 compared with the control group, and "**" indicates P < 0.01 compared with the control group.

[0058] Example 2

[0059] Preparation method of the compound described in the present invention:

[0060] (1) Take 50 kg of Abelmoschus manihot flowers and extract with 500 L of 70% ethanol, and concentrate the extract until the alcohol smell disappears;

[0061] (2) The concentrated solution is successively extracted 3 times with equal volumes of petroleum ether, dichloromethane, and ethyl acetate. The extracts are concentrated under reduced pressure to obtain extracts, namely petroleum ether extract, dichloromethane extract, and ethyl acetate extract;

[0062] (3) Perform silica gel column chromatography on the ethyl acetate extract, with the ratio of medicinal material to silica gel being 1:10 (W / W), elute with a mixed solution of ethyl acetate and ethanol with a volume ratio of 25:1 for 5 column volumes, discard the eluate, and then elute with a mixed solution of ethyl acetate and ethanol with a volume ratio of 15:1 for 13 column volumes, collect the eluate, and concentrate to an extract;

[0063] (4) Dissolve the extract in methanol, further separate it by Sephadex LH-20 column chromatography, with the diameter-to-height ratio being 1:100, use 75% ethanol solution as the mobile phase, collect the orange band, recover the solvent, and combine the relatively pure fractions under the guidance of TLC. Repeat this step for purification until the purity detected by high-performance liquid chromatography reaches more than 98%.

[0064] HPLC conditions:

[0065] Chromatographic column: C18 column

[0066] Mobile phase: methanol-0.1% phosphoric acid solution (64:36)

[0067] Detection wavelength: 330nm

[0068] Column temperature: 30°C

[0069] Injection volume: 10 μl

[0070] Example 3

[0071] Structural confirmation of the compounds described in the present invention

[0072] Orange-yellow amorphous powder, soluble in methanol. High-resolution mass spectrometry yielded m / z 447.0719 [M+H] + (calculated value 447.0716) molecular ion peak, combined with 1 H-NMR spectra and 13 The molecular formula of the compound was inferred to be C 24 H 14 O9. 1 In the H-NMR spectrum, δ6.28 (d, 1H, J = 5.7 Hz, H-2”), δ8.18 (d, 1H, J = 5.7 Hz, H-3”) are the double bond proton signals of γ-pyrone, δ6.32 (d, 1H, J = 1.6 Hz, H-5), δ6.39 (d, 1H, J = 1.6 Hz, H-7) are the meta-coupled benzene ring proton signals, δ6.82 (d, 1H, J = 8.2 Hz, H-5’), δ7.21 (dd, 1H, J=2.0,8.4Hz,H-6'),δ7.42(d,1H,J=2.0Hz,H-2') are ABX coupling proton signals of benzene ring,δ6.36(s,1H,H-6”),δ6.51(s,1H,H-10) are aromatic proton signals,δ9.24(s,1H),δ9.61(s,1H),δ10.89(s,1H),δ11.12(s,1H),δ12.70(s,1H) are hydroxyl proton signals. 13 In the C-NMR spectrum, a total of 24 carbon signals were given. Combined with the HSQC spectrum and the HMBC spectrum, the relevant carbon and hydrogen signals were assigned, as shown in Table 5. Based on the above information, the chemical structure of the compound described in the present invention is determined to be:

[0073]

[0074] Table 5 1 H(600MHz)and13 C(150 MHz) NMR Spectroscopic Data in DMSO (δ in ppm, J in Hz).

[0075]

[0076]

Claims

1. A compound, characterized in that, The chemical structure is as follows:

2. The compound according to claim 1, wherein, The preparation method comprises the following steps: (1) Extract the Flos Abelmoschus manihot with 70% ethanol, and concentrate the extract until the alcohol smell disappears; (2) Extract the concentrated solution successively with petroleum ether, dichloromethane, and ethyl acetate. The extracts are concentrated under reduced pressure to obtain extracts, namely petroleum ether extract, dichloromethane extract, and ethyl acetate extract respectively; (3) Further separate the ethyl acetate extract by silica gel column chromatography, elute with a mixed solution of ethyl acetate and ethanol with a volume ratio of 25:1, discard the eluate, then elute with a mixed solution of ethyl acetate and ethanol with a volume ratio of 15:1, collect the eluate, and concentrate it to an extract; (4) Dissolve the extract in methanol, further purify it by Sephadex LH-20 column chromatography, use 75% ethanol solution as the mobile phase, collect the orange band, recover the solvent, and repeat this step repeatedly until the purity detected by high performance liquid chromatography reaches over 98%; 3. The compound according to claim 1, wherein It can significantly reduce the rates of soft eggshells and broken eggshells.

4. The compound according to claim 1, wherein It is used for preventing and treating soft-shelled eggs and broken eggs in poultry.

5. The compound according to claim 1, wherein It forms a composition with quercetin.

6. The composition according to claim 5, wherein It comprises 0.05 - 0.2 parts of the compound described in claim 1 and 1 part of quercetin.

7. The composition according to claim 5, wherein It comprises 0.1 part of the compound and 1 part of quercetin.

8. The composition according to claim 5, wherein It is used for preventing and treating soft-shelled eggs and broken eggs in poultry while improving the egg-laying performance of poultry.

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