Use of a compound and compositions thereof in the control of soft and broken shell eggs in poultry

Abstract

The application discloses a compound and a use of a composition of the compound in preventing and treating soft-shelled eggs and broken-shelled eggs of poultry, and a chemical structural formula of the compound is shown in the figure.The compound is prepared by extracting Malva sylvestris flowers and then separating and purifying the Malva sylvestris flowers.The compound can significantly reduce the rate of soft-shelled eggs and broken-shelled eggs.A composition of the compound and quercetin can significantly reduce the rate of soft-shelled eggs and broken-shelled eggs, obviously improve the daily egg production rate, reduce the feed-egg ratio, improve the egg production performance of poultry, and improve the economic benefits of poultry farmers.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of veterinary drugs, and particularly relates to a compound and a composition thereof for preventing and treating soft-shelled eggs and broken-shelled eggs produced by poultry. BACKGROUND

[0002] At present, the food safety problem in China is quite serious. Due to the abuse of veterinary drugs, antibiotics, hormones and other drugs are accumulated in poultry, which further leads to the drug residue in eggs produced by poultry exceeding the standard, and seriously affects the quality of egg products. In recent years, the veterinary drugs obtained from traditional Chinese medicines, which are pure natural, non-antibiotic hormones and can improve the egg production performance of poultry, have become a research hotspot. However, compared with the large number of traditional Chinese medicines, only a small number of traditional Chinese medicines are used in veterinary drugs, and it is of great significance to explore traditional Chinese medicines with potential for veterinary drugs.

[0003] After the peak egg production period of poultry, with the increase of age, the physiological function gradually declines, especially in the later egg production period, the egg production rate decreases, the eggshell quality deteriorates, the soft-shelled egg and broken-shelled egg rate significantly increases, and at the same time, the feed intake of poultry increases, and the economic benefit decreases.

[0004] Flavonoids are polyphenols with C6-C3-C6 structure existing in plants, and have various physiological functions, and have wide application value in the fields of medicine, food and health care products. Flavonoids can improve the reproductive ability of animals by affecting the hormones related to reproduction in livestock and poultry. In the prior art, there are reports that adding soybean isoflavones, quercetin and other flavonoids and curcumin, gallnut tannin and other polyphenols in feed can improve the production performance of poultry and the quality of eggs.

[0005] Malva sylvestris L. Hibiseu manihot , also known as Malva sylvestris L., is an annual herbaceous plant of Malvaceae Malva, and is rich in flavonoids in its flowers, which is an ideal medicinal part. SUMMARY

[0006] In view of the deficiencies of the prior art, the purpose of the present application is to prepare a compound and a composition thereof, and to develop the compound and the composition thereof as a veterinary drug for preventing and treating soft-shelled eggs and broken-shelled eggs produced by poultry and improving the egg production performance of poultry.

[0007] The compound according to the present application has the following chemical structure:

[0008] .

[0009] The preparation method of the compound according to the present application comprises the following steps:

[0010] (1) Malva sylvestris L. flowers are extracted with 70% ethanol, and the extract is concentrated to be alcohol-free;

[0011] (2) The concentrated liquid is extracted with petroleum ether, dichloromethane and ethyl acetate in sequence, and the extracted liquid is concentrated into an extract under reduced pressure to obtain petroleum ether extract, dichloromethane extract and ethyl acetate extract, respectively;

[0012] (3) The ethyl acetate extract is further separated by silica gel column chromatography, eluted with a mixture of ethyl acetate and ethanol in a volume ratio of 25:1, discarded, and then eluted with a mixture of ethyl acetate and ethanol in a volume ratio of 15:1, collected and concentrated into an extract;

[0013] (4) The extract is dissolved in methanol and further purified by Sephadex LH-20 column chromatography, using 75% ethanol solution as the mobile phase, collecting orange color bands, and repeatedly repeating the step until the purity reaches more than 98% by high performance liquid chromatography.

[0014] The compound can significantly reduce the rate of soft eggshell and broken eggshell, and is used for preventing and treating soft eggshell and broken eggshell of poultry.

[0015] The composition comprises the compound and quercetin. The proportion of the composition is 0.05-0.2 parts of the compound and 1 part of quercetin, and preferably the proportion is 0.1 part of the compound and 1 part of quercetin.

[0016] The composition can improve the egg-laying performance of poultry while preventing and treating soft eggshell and broken eggshell of poultry. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 NMR of the compound 1 H-NMR spectrum

[0018] Figure 2 NMR of the compound 13 C-NMR spectrum

[0019] Figure 3 HSQC spectrum of NMR of the compound

[0020] Figure 4 HMBC spectrum of NMR of the compound DETAILED DESCRIPTION

[0021] The application will be further described below in combination with examples, but it does not limit the implementation of the application.

[0022] Example 1: Effect of the compound and the composition of the compound and quercetin on the egg-laying performance of Hyline brown egg-laying hens in the later stage

[0023] 1.1 Experimental animals: 500 Hy-Line Brown laying hens of 55 weeks 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 1 group (compound 0.05 parts, quercetin 1 part), composition 2 group (compound 0.1 part, quercetin 1 part), and composition 3 group (compound 0.2 part, quercetin 1 part), as shown in Table 1. Each group had 5 replicates, each replicate had 10 chickens, and the pre-test period was 3 weeks, during which the chickens were fed with the basic feed, as shown in Table 2. The test period was 8 weeks in total.

[0024] Table 1 Experimental design

[0025]

[0026] Table 2 Composition and nutritional level of the basic feed

[0027]

[0028] a The premix provided VA 9000 IU, VD 2500 IU, VE 20 IU, VB 1212 μg, VK 2.4 mg per kg of feed; trace elements Mn 100 mg, Zn 60 mg, Fe 25 mg, Cu 10 mg, Se (N2SeO3·5H2O) 0.35 mg, I (KI) 0.5 mg.

[0029] 1.2 Reagents and main instruments: The estradiol detection kit and the progesterone detection kit were purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd.

[0030] Analytical balance (Mettler), table centrifuge (Shanghai Anting), constant temperature water bath, Multiskan FC enzyme label instrument (Sanofi).

[0031] 1.3 Determination method

[0032] 1.3.1 The determination method of each index of production performance is as follows:

[0033] (1) Daily egg laying rate = total number of eggs in the statistical period ÷ (feeding days × number of laying hens) × 100%

[0034] (2) Average egg weight = total egg weight ÷ total number of eggs

[0035] (3) Daily feed intake = total feed consumption in the statistical period ÷ (feeding days × number of laying hens)

[0036] (4) Feed-egg ratio (feed conversion rate) = total feed consumption in the statistical period ÷ total egg weight.

[0037] (5) Soft-shelled egg and broken-shelled egg rate = number of soft-shelled eggs and broken-shelled eggs ÷ total number of eggs × 100%

[0038] 1.3.2 Endocrine hormone index determination

[0039] After the end of the feeding experiment, one hen was randomly selected from each group, blood was collected from the anterior vena cava, serum was separated and stored in a refrigerator at -30℃.

[0040] The serum estradiol and progesterone were determined by enzyme-linked immunoassay.

[0041] 1.4 The experimental data were processed by SPSS20.0 software, all data were statistically analyzed and expressed as (mean ± s), and the comparison among multiple groups was analyzed by One-Way-ANOVA method. P<0.05 indicates a statistically significant difference.

[0042] 1.5 Index detection and experimental results

[0043] 1.5.1 Production performance

[0044] Compared with the control group, the addition of the compound in the feed can reduce the rate of soft-shelled eggs and broken-shelled eggs, and with the increase of the addition level, the rate of soft-shelled eggs and broken-shelled eggs is significantly reduced, and the difference is statistically significant; the addition of the compound in the feed can improve the egg production rate, and the difference is statistically significant; the addition of the compound in the feed has no significant effect on the average egg weight, daily feed intake and feed egg ratio.

[0045] Compared with the control group, the addition of quercetin in the feed can improve the daily egg production rate, and with the increase of the addition level, the daily egg production rate is significantly improved, and the difference is statistically significant; the addition of quercetin in the feed can reduce the feed egg ratio, and the difference is statistically significant; the addition of quercetin in the feed has no significant effect on the average egg weight, daily feed intake, soft-shelled egg rate and broken-shelled egg rate.

[0046] Compared with the control group, the addition of the combination of the compound and quercetin in the feed can improve the daily egg production rate, and the optimal combination ratio is 0.1 part of the compound and 1 part of quercetin, and the difference is statistically significant; the addition of the combination of the compound and quercetin in the feed can reduce the rate of soft-shelled eggs and broken-shelled eggs, and the optimal combination ratio is 0.1 part of the compound and 1 part of quercetin, and the difference is statistically significant; the addition of the combination of the compound and quercetin in the feed can reduce the feed egg ratio, and the difference is statistically significant; the addition of the combination of the compound and quercetin in the feed has no significant effect on the average egg weight and daily feed intake.

[0047] See Table 3 for details.

[0048] Table 3 Effect of the compound, quercetin and their combination according to the application on the egg production performance of Hyline brown shell laying hens in the later stage of egg production

[0049] ​

[0050] Note: "*" indicates P < 0.05 compared with the control group, "**" indicates P < 0.01 compared with the control group.

[0051] 1.5.2 Serum hormone determination

[0052] Compared with the control group, the addition of the compound in the feed can significantly improve the serum estradiol and progesterone levels of Hyline brown shell laying hens in the later laying period, and the difference is statistically significant; the addition of quercetin in the feed can significantly improve the serum estradiol and progesterone levels of Hyline brown shell laying hens in the later laying period, and the difference is statistically significant; the combination of 0.1 parts of the compound and 1 part of quercetin has the optimal effect on improving the estradiol and progesterone levels, as shown in Table 4.

[0053] Table 4 Effect of the compound, quercetin and their combination on serum hormone levels of Hyline brown shell laying hens in the later laying period

[0054]

[0055] Note: "*" indicates P < 0.05 compared with the control group, "**" indicates P < 0.01 compared with the control group.

[0056] Example 2: Preparation method of the compound of the present application:

[0057] (1) Take 50 kg of Hibiscus sabdariffa flowers and extract with 500 L of 70% ethanol, and concentrate the extract to remove the alcohol taste;

[0058] (2) The concentrated liquid is successively extracted with an equal volume of petroleum ether, dichloromethane and ethyl acetate for 3 times, and the extract is concentrated into an extract under reduced pressure to obtain petroleum ether extract, dichloromethane extract and ethyl acetate extract, respectively;

[0059] (3) The ethyl acetate extract is subjected to silica gel column chromatography, and the medicinal material:silica gel = 1:10 (W / W), eluted with a mixture of ethyl acetate and ethanol with a volume ratio of 25:1 for 5 column volumes, and then eluted with a mixture of ethyl acetate and ethanol with a volume ratio of 15:1 for 13 column volumes, and the eluate is collected and concentrated into an extract;

[0060] (4) The extract is dissolved with methanol and further separated by Sephadex LH-20 column chromatography, with a diameter-height ratio of 1:100, and 75% ethanol solution as the mobile phase, and the orange color band is collected, the solvent is recovered, and the relatively pure fractions are combined under the guidance of TLC, and this step is repeated repeatedly until the purity reaches more than 98% by high performance liquid chromatography detection.

[0061] High performance liquid chromatography conditions:

[0062] Chromatographic column: C18 chromatographic column

[0063] Flow direction: methanol-0.1% phosphoric acid solution (64:36) as mobile phase

[0064] Detection wavelength: 330 nm

[0065] Column temperature: 30°C

[0066] Injection volume: 10 μl

[0067] Example 3: Structural confirmation of the compound of the present application

[0068] Orange yellow amorphous powder, soluble in methanol. High resolution mass spectrum gave m / z 447.0719 [M + H] + a molecular ion peak of 447.0719 [M + H] (calcd 447.0716), combined with 1 H-NMR spectrum and 13 C-NMR spectrum data deduced the molecular formula of the compound as C 24 H 14 O9. In 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’’) were 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) were meta-coupled benzene ring proton signals, δ6.82 (d, 1H, J = 8.2 Hz, H-5’), δ7.21 (dd, 1H, J = 2.0, 8.4 Hz, H-6’), δ7.42 (d, 1H, J = 2.0 Hz, H-2’) were ABX-coupled benzene ring proton signals, δ6.36 (s, 1H, H-6’’), δ6.51 (s, 1H, H-10) were aromatic proton signals, δ9.24 (s, 1H), δ9.61 (s, 1H), δ10.89 (s, 1H), δ11.12 (s, 1H), δ12.70 (s, 1H) were hydroxyl proton signals. In 13 In the C-NMR spectrum, 24 carbon signals were given, and the relevant carbon and hydrogen signals were assigned by combining HSQC spectrum and HMBC spectrum, see Table 5. Based on the above information, the chemical structure of the compound of the present application was determined as:

[0069]

[0070] Table 5 1 H (600 MHz) and 13C (150 MHz) NMR Spectroscopic Data in DMSO ( delta in ppm, J in Hz).

[0071] .

Claims

1. A compound, characterized in that, The chemical structure is as follows: 。 2. A process for the preparation of a compound according to claim 1, characterized in that, The method comprises the following steps: (1) Malve flower is extracted with 70% ethanol, and the extract is concentrated to remove alcohol; (2) The concentrated solution is extracted with petroleum ether, dichloromethane and ethyl acetate in sequence, and the extract is concentrated into extract paste under reduced pressure to obtain petroleum ether extract, dichloromethane extract and ethyl acetate extract; (3) The ethyl acetate extract is further separated by silica gel column chromatography, eluted with a mixture of ethyl acetate and ethanol with a volume ratio of 25:1, discarded, and then eluted with a mixture of ethyl acetate and ethanol with a volume ratio of 15:1, collected and concentrated into extract paste; (4) The extract paste is dissolved in methanol and further purified by Sephadex LH-20 column chromatography, and the orange band is collected with 75% ethanol solution as the mobile phase; the solvent is recovered, and the step is repeated repeatedly until the purity detected by high performance liquid chromatography reaches more than 98%.

3. Use of a compound according to claim 1, characterized in that, The soft eggshell and broken eggshell rate can be significantly reduced.

4. Use of a compound according to claim 1, characterized in that, The method is used for preventing and treating soft eggshell and broken eggshell of poultry.

5. A composition consisting of the compound of claim 1 and quercetin.

6. The composition of claim 5, wherein, The composition consists of 0.05-0.2 parts of the compound of claim 1 and 1 part of quercetin.

7. The composition of claim 5, wherein, The composition consists of 0.1 part of the compound of claim 1 and 1 part of quercetin.

8. The composition of claim 5, wherein, The method is used for preventing and treating soft eggshell and broken eggshell of poultry and improving the egg-laying performance of poultry.

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