A composite plant extract and its application in resisting heat stress in poultry

Abstract

The invention belongs to the technical field of poultry breeding, and in particular relates to a composite plant extract and an application thereof in preventing heat stress in poultry. The composite plant extract is prepared by mixing quercetin, rutin, and a Prunella vulgaris extract in a mass ratio of 3:3:1. The purity of the quercetin is 98%, and the purity of the rutin is 95%. The Prunella vulgaris extract is prepared by the following method: decocting the Prunella vulgaris herb with water 1-3 times for 2-3 hours each time, filtering, concentrating the decoction, adding 70-90% ethanol to adjust the alcohol content to 55-60%, allowing the decoction to stand, collecting the supernatant, and concentrating to obtain an extract. The composite plant extract composed of the three plant extracts can be used for preventing and treating heat stress in poultry.

Description

Technical Field

[0001] The invention belongs to the technical field of poultry breeding, and in particular relates to a composite plant extract and application thereof in resisting heat stress. Background Art

[0002] The continued global warming in recent years has posed significant challenges to poultry farming worldwide. Oxidative stress, caused by high temperatures, high humidity, and intensive farming, has become one of the major hazards in poultry farming. Oxidative stress, defined as an imbalance in the redox state of cells or organisms, can reduce poultry production performance and meat quality, and increase mortality. In poultry production, stressors can arise from factors such as feed, rearing conditions, and the environment. Nutritionally, food intake, digestion, and intestinal absorption of nutrients can generate ROS and free radicals in the intestinal mucosa, liver, and peripheral metabolic sites, leading to an imbalance in the antioxidant system, lipid peroxidation, and damage to membranes and other biomolecules (including DNA). The liver is more susceptible to oxidative stress than other organs. As a major detoxification and anabolic organ, it produces free radicals when excessive fat, metabolites, and toxins accumulate in the liver. To alleviate oxidative stress in broiler chickens, the addition of antioxidants to broiler diets has become a research hotspot. Natural plant extracts are considered to be one of the most promising natural antioxidants in various animal diets due to their unique advantages such as naturalness, versatility, low toxicity and side effects, and no residue. They can be used to alleviate the oxidative stress suffered by broiler chickens during production. Summary of the Invention

[0003] The purpose of the present invention is to provide a composite plant extract and application thereof.

[0004] In order to achieve the above object, the technical solution adopted by the present invention is:

[0005] A composite plant extract is prepared by mixing quercetin, rutin and selfheal extract in a mass ratio of 3:3:1. The purity of quercetin is 98%, and the purity of rutin is 95%. The selfheal extract is prepared by the following method: selfheal herbs are decocted with water 1-3 times, each time for 2-3 hours, filtered, the decoction is concentrated, 70-90% ethanol is added to make the alcohol content 55-60%, the decoction is allowed to stand, the supernatant is collected, and concentrated to obtain the extract.

[0006] The invention discloses an application of a composite plant extract, wherein the composite plant extract has strong in vitro antioxidant capacity.

[0007] The invention discloses an application of a composite plant extract, which can alleviate the toxicity of chicken embryo primary hepatocytes induced by hydrogen peroxide at the cellular level.

[0008] The invention discloses an application of a composite plant extract. The composite plant extract is prepared by mixing 300g of rutin extract, 300g of quercetin extract and 100g of Prunella vulgaris extract with the remainder being anhydrous glucose to prepare 1kg of the composite plant extract. The composite plant extract is used as a functional feed additive in broiler feed. When added at a dosage of 1kg / ton, the composite plant extract is used to treat problems such as poor growth and development, low nutrient metabolism rate and low serum antioxidant enzyme activity caused by heat stress in broiler chickens.

[0009] The invention discloses an application of a composite plant extract, wherein the composite plant extract is added to feed to alleviate heat stress in poultry.

[0010] The present invention has the following advantages: Rutin is a flavonoid compound widely found in Chinese herbal medicines, fruits, and vegetables, and possesses a wide range of pharmacological activities, including anti-inflammatory, antioxidant, and antiviral effects. Studies have shown that rutin can scavenge free radicals in the human body, inhibit the growth of Staphylococcus aureus in food, and also has anti-cancer effects. Rutin also reduces the formation of sorbitol, reactive oxygen species, precursors of advanced glycation end products, and inflammatory cytokines. These effects are believed to explain rutin's protective effects against nephropathy, neuropathy, liver damage, and cardiovascular diseases caused by hyperglycemia and dyslipidemia. Quercetin is an important flavonoid substance abundant in fruits and vegetables. In vitro, it exhibits potent antioxidant capacity by directly scavenging reactive oxygen free radicals, chelating metal ions, inhibiting lipid peroxidation, and inhibiting DNA oxidative damage. In vivo, it exerts its antioxidant effects primarily by protecting vascular endothelial cells, increasing nitric oxide levels, and increasing the total antioxidant capacity of peripheral blood. It also possesses multiple biological activities and pharmacological effects, including anti-inflammatory and anti-cancer effects. Prunella vulgaris is a common Chinese medicinal herb with antibacterial and anti-inflammatory properties, and can be used to treat infectious diseases. Prunella vulgaris can inhibit the growth of pathogenic microorganisms and alleviate inflammatory responses. Based on these three extracts, the present invention screened a composite plant extract based on its in vitro antioxidant capacity. The composite plant extract's protective effect against oxidative damage was validated in experiments with primary chicken embryo hepatocytes, and finally verified in a 21-day broiler heat stress experiment. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is a standard curve diagram of 1-6 mmol / LFeSO4 in Experimental Example 1 of the present invention. DETAILED DESCRIPTION

[0012] Example

[0013] A composite plant extract is prepared by mixing quercetin, rutin and selfheal extract in a mass ratio of 3:3:1. The purity of quercetin is 98%, and the purity of rutin is 95%. The selfheal extract is prepared by the following method: selfheal herbs are decocted with water 1-3 times, each time for 2-3 hours, filtered, the decoction is concentrated, 70-90% ethanol is added to make the alcohol content 55-60%, the decoction is allowed to stand, the supernatant is collected, and concentrated to obtain the extract.

[0014] The invention discloses an application of a composite plant extract, which can alleviate the toxicity of chicken embryo primary hepatocytes induced by hydrogen peroxide at the cellular level.

[0015] The invention discloses an application of a composite plant extract, wherein the composite plant extract is added to broiler feed as an additive for preparing a functional feed for treating poor growth and development, low nutrient metabolism rate and low serum antioxidant enzyme activity caused by heat stress in broiler chickens.

[0016] The invention discloses an application of a composite plant extract, wherein the composite plant extract is added to feed to alleviate oxidative stress in poultry farming.

[0017] Experimental example

[0018] Experiment 1 Screening of compound plants extract (CPE)

[0019] 1 Materials and Methods

[0020] 1.1 Experimental Materials

[0021] 95% rutin (purchased from Shanghai Shifeng Biotechnology Co., Ltd.); 98% quercetin (purchased from Shaanxi Shengqing Biotechnology Co., Ltd.); Prunella vulgaris (purchased from Queshan); DDPH (purchased from Bestone Lab Supplies Mall); ABTS (purchased from Bestone Lab Supplies Mall); potassium peroxydisulfate (purchased from Dongguan Xilong Scientific Guangzhou Fanyu Liqiang); TPTZ (purchased from Chemical Reagent City); PTIO (purchased from Hefei Yika Biological Reagent); vitamin C (purchased from Xilong Scientific Co., Ltd.); anhydrous sodium acetate (purchased from Rongfengxian Lab Supplies Mall Head Office); glacial acetic acid (≥99.0% g / g, purchased from standard laboratory analytical pure chemical reagents); FeSO4 (purchased from Jiehui Chemical); vitamin C (≥99.7%, Shanghai Zhanyun Biochemical Co., Ltd.).

[0022] 1.2 Preparation of Prunella Vulgaris Extract

[0023] Decoction of Prunella vulgaris root is performed 1-3 times, each time for 2-3 hours. The decoction is filtered and concentrated. 70-90% ethanol is added to reduce the alcohol content to 55-60%. The mixture is allowed to stand. The supernatant is collected and concentrated to obtain an extract. 1g of Prunella vulgaris extract is obtained from 20g of Prunella vulgaris root.

[0024] 1.3 Preparation of rutin, quercetin and prunella vulgaris solution

[0025] 0.5 g each of rutin, quercetin and Prunella vulgaris extract were dissolved in 10 mL of methanol, filtered three times with qualitative filter paper, and then diluted to 100 mL with deionized water. The solution was filtered with a 0.22 μm filter membrane and stored at 4 °C for later use.

[0026] 1.4 Preparation of Vitamin C Solution

[0027] Dissolve 10 mg of vitamin C in 100 mL of deionized water, filter through a 0.22 μm filter membrane, and store at 4°C until use.

[0028] 1.5 Evaluation of antioxidant properties

[0029] PTIO Free Radical Scavenging Test: Dissolve 30 mg of PTIO solid in 200 mg of distilled water and shake thoroughly to dissolve it completely, thereby obtaining a PTIO working solution. Next, add varying volumes of the sample solution to a reaction tube. To evaluate the scavenging effect of PTIO, measure the absorbance of the solution at a wavelength of 557 nm, denoted as A. Simultaneously, as a control, measure the absorbance under the same conditions without the addition of sample, denoted as A0. The value of A0 should be between 0.2 and 0.6 to ensure experimental accuracy. Finally, calculate the PTIO scavenging effect according to the following formula: PTIO Scavenging Effect = (A0 - A) / A0 × 100%, where A0 represents the value without the addition of sample.

[0030] ABTS·+ free radical scavenging test:

[0031] Mix 0.2 mg of ABTS diammonium salt stock solution and 0.2 mg of K2S2O8 stock solution in a 1:1 ratio. The mixture is then placed in the dark at room temperature for 12 hours to allow ABTS and K2S2O8 to fully react and generate ABTS·+ free radicals. The mixture is gradually diluted with anhydrous ethanol at pH 7.4 until its absorbance reaches a range of 0.70±0.02. This diluted solution is the ABTS·+ working solution. Add different volumes of the sample solution to the reaction tube. Allow the sample to react with the ABTS·+ working solution at room temperature for 6 minutes. To evaluate the sample's ABTS free radical scavenging ability, measure the absorbance of the post-reaction solution at a wavelength of 734 nm, denoted as A. Simultaneously, as a control, measure the absorbance without the addition of the sample under the same conditions, denoted as A0. The ABTS scavenging effect of the sample was calculated according to the following formula: ABTS scavenging effect = (A0-A) / A0×100%, where A represents the absorbance of the reaction solution after adding different volumes of sample.

[0032] DPPH Free Radical Scavenging Test: Dissolve 3 mg of solid DPPH in 72 mL of anhydrous ethanol. Shake thoroughly to ensure that the solid DPPH is completely dissolved in the anhydrous ethanol. The prepared DPPH solution must be stored away from light and used within five hours to ensure its stability and accuracy. Dilute 72 mL of the prepared DPPH solution with 36 mL of anhydrous ethanol. Adjust the absorbance of the solution to a range of 0.6 to 1.0 by gradually adding anhydrous ethanol. If the absorbance of the solution is too high, continue to add anhydrous ethanol for dilution. If the absorbance is too low, add more solid DPPH. This adjustment results in the DPPH working solution. Add varying volumes of the sample solution to the reaction tube. Allow the sample to react with the DPPH working solution at room temperature for 30 minutes. After the reaction, the absorbance of the solution was measured at a wavelength of 519 nm using a spectrophotometer, recorded as A. For comparison, the absorbance of the DPPH working solution at 519 nm was measured without adding any sample, recorded as A0. Finally, the DPPH scavenging effect of the sample was calculated using the following formula: DPPH scavenging effect = (A0 - A0) / A0 × 100%.

[0033] FRAP total antioxidant capacity assay: When performing the antioxidant capacity assay, 0.1 mL of sample solution was injected into the reaction tube, and then 2.4 mL of FRAP (Ferric Reducing Antioxidant Power) working solution was added. After the two were thoroughly mixed, the reaction tube was placed in a 37°C water bath and heated for 10 minutes. The absorbance of the solution was measured at a wavelength of 593 nm using a spectrophotometer. In order to accurately calculate the antioxidant capacity of the sample, a standard curve was drawn using a 1-6 mmol / L FeSO4 standard solution ( Figure 1 ). Through this standard curve, the regression equation Y = 0.0692x + 0.0247 was obtained, and its correlation coefficient R 2 The linear relationship is 0.971. Substituting the measured sample absorbance into this regression equation, the sample's FRAP value is calculated. One FRAP unit is defined as the antioxidant capacity equivalent to 1 mmol / L of FeSO₄. This method directly determines the antioxidant capacity of a sample relative to the amount of FeSO₄ present.

[0034] 1.6 Screening of compound plant extracts (CPE)

[0035] A three-factor, three-level orthogonal experiment was conducted on the composite plant extracts, with the three extracts as the three factors and the three plant extracts at 1, 2, and 3 parts by weight, respectively. The design table is shown in Table 1.

[0036] Table 1 Orthogonal experimental design table

[0037] Quercetin Rutin Prunella vulgaris 1 1 1 1 2 1 2 2 3 1 3 3 4 2 1 2 5 2 2 3 6 2 3 1 7 3 1 3 8 3 2 1 9 3 3 2

[0038] 2 Results and Analysis

[0039] 2.1 Evaluation of antioxidant properties of different herbs

[0040] Table 2 shows an evaluation of the PTIO free radical scavenging effects of the three herbs. Rutin demonstrated the greatest PTIO free radical scavenging activity, equivalent to 469.37 mgVc / g, compared to 318.51 mgVc / g for quercetin and 100.55 mgVc / g for Prunella vulgaris, with significant differences (P > 0.05). In the PTIO free radical scavenging assay, the antioxidant capacity of the three substances ranked in the order of rutin > quercetin > Prunella vulgaris.

[0041] Table 2 PTIO free radical scavenging effect

[0042] extract concentration Sample volume (μL) Clearance rate (%) Vc (mg / g) Rutin 0.5g / 100mL 100 37.39±2.28 469.37±23.1A Quercetin 0.5g / 100mL 100 25.37±1.72 318.51±18.00B Prunella vulgaris 0.5g / 100mL 500 40.05±3.37 100.55±7.49C Vitamin C 0.1g / 100mL 400 63.72±3.67

[0043] The same capital letters in the same column indicate no significant difference (P>0.05); different capital letters indicate significant difference (P<0.05), the same below.

[0044] Table 3 evaluates the DPPH free radical scavenging effects of the three substances. Table 2 shows that quercetin exhibits the highest DPPH free radical scavenging activity, equivalent to 82.61 mgVc / g. Rutin exhibits no significant difference (P < 0.05) at 53.15 mgVc / g, while Prunella vulgaris exhibits a significant difference (P > 0.05) at 49.38 mgVc / g. Therefore, in the DPPH free radical scavenging test, the antioxidant capacity of the three substances is quercetin > rutin > Prunella vulgaris.

[0045] Table 3 DPPH free radical scavenging effect

[0046] extract concentration Sample volume (μL) Clearance rate (%) Vc (mg / g) Rutin 0.5g / 100mL 60 53.15±3.62 118.46±8.07B Quercetin 0.5g / 100mL 60 72.61±3.90 161.83±8.69A Prunella vulgaris 0.5g / 100mL 100 49.38±1.18 66.9±0.16C Vitamin C 0.1g / 100mL 50 74.71±5.01

[0047] Table 4 shows an evaluation of the ABTS·+ free radical scavenging effects of the three substances. Table 3 shows that quercetin had the greatest ABTS·+ scavenging effect, equivalent to 106.91 mgVc / g. Rutin had a similar effect (101.14 mgVc / g) with no significant difference (P < 0.05). Prunella vulgaris had a significantly different effect (75.68 mgVc / g) (P > 0.05). Therefore, in the ABTS·+ free radical scavenging test, the antioxidant capacity of the three substances ranked in the order of quercetin > Prunella vulgaris > rutin.

[0048] Table 4 ABTS + Free radical scavenging effect

[0049] extract concentration Sample volume (μL) Clearance rate (%) Vc (mg / g) Rutin 0.5g / 100mL 100 67.14±8.71 38.43±2.00C Quercetin 0.5g / 100mL 100 93.91±4.75 53.75±2.74A Prunella vulgaris 0.5g / 100mL 500 80.68±6.59 46.18±0.75B Vitamin C 0.1g / 100mL 15 52.41±5.60

[0050] The evaluation of the FRAP values ​​of the three substances is shown in Table 5. As shown in Table 5, quercetin has the largest FRAP value of 1.45 mmol / LFeSO4, which is significantly different from rutin (1.01 mmol / L FeSO4) (P>0.05). Prunella vulgaris has a FRAP value of 0.93 mmol / LFeSO4. The FRAP values ​​of the three samples are ranked as follows: quercetin > rutin > Prunella vulgaris.

[0051] Table 5 FRAP value experimental results

[0052] extract concentration Sample volume (μL) FRAP Vc (mg / g) Rutin 0.5g / 100mL 100 1.05 0.48±0.03B Quercetin 0.5g / 100mL 100 1.45 0.66±0.05A Prunella vulgaris 0.5g / 100mL 100 0.93 0.43±0.04C Vitamin C 0.1g / 100mL 500 2.18

[0053] The results of the orthogonal test of the three substances are shown in Table 6. As can be seen from this table, the main effect of the three substances combined was quercetin > rutin > prunella vulgaris. Theoretical analysis of the orthogonal experiment indicated that the total antioxidant capacity was maximized when the three substances were combined in a ratio of 3:3:1. Repeated verification of this ratio yielded ABTS, DPPH, PTIO, and FRAP values ​​of 59.67, 173.69, 321.59, and 0.89 VC mg / g, respectively, for a total antioxidant capacity of 555.84 VC mg / g. Based on this comprehensive analysis, this optimal ratio was used for subsequent experiments.

[0054] Table 6 Orthogonal experiment results

[0055]

[0056] Experiment 2: Study on the effect of compound plants extract (CPE) on alleviating hydrogen peroxide-induced toxicity in primary chicken embryo hepatocytes

[0057] 1 Materials and Methods

[0058] 1.1 Drugs and reagents

[0059] A complex plant extract (quercetin:rutin:Prunella vulgaris = 3:3:1) was dissolved in anhydrous ethanol. DMEM / F-12 medium (modified by mixing DMEM and Ham's F-12 medium at a 1:1 ratio), fetal bovine serum (FBS), and trypsin-EDTA were purchased from Gibco (Carlsbad, CA, USA). Penicillin-streptomycin antibiotic solution was purchased from Pricells (Wuhan, China). Phosphate-buffered saline (PBS) was purchased from Dulbecco's. ML385 was purchased from GlpBio.

[0060] 1.2 Cell preparation and treatment

[0061] The culture medium of CEHs cells consisted of high glucose Dulbecco's modified Eagle's medium (DMEM) / F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.

[0062] Get the sterile chicken embryo of 14 embryonic ages and be positioned in the disposable sterile culture dish, decapitation is put to death rapidly and open the abdomen and take out liver and place 4 ℃ of precooling phosphate buffered saline (PBS) that contains 1% penicillin-streptomycin, peel off after being placed in respectively 5mL centrifuge tube and shear, add 2mL 0.25% trypsin, place 37 ℃ of water-baths and digest for 30min, shake 1 time every 3min, treat that bulk liver tissue disappears after adding the DMEM / F12 culture medium that 2mL contains 10% fetal bovine serum and 1% penicillin-streptomycin and mix, filter and collect filtrate with 200 mesh cell sieves, with centrifugal 10min of 1000r / min, remove supernatant and add the DMEM / F12 complete medium of 1.5ml and the 3mL 50%Percoll cell separation fluid of preparing in advance and mix, with the speed centrifugal 15min of 3000r / min. After centrifugation, take 2 mL of the middle layer and place it in a new centrifuge tube. Add an equal amount of 2 mL of DMEM / F12 complete medium and centrifuge at 1000 r / min for 10 min. Discard the supernatant and add DMEM / F12 complete medium to resuspend the hepatocytes. Then, add 1.0×10 5 Cells were seeded into 6-well or 96-well plates at a density of 100 cells / mL. The culture medium was replaced every 48 h. Cells were observed and photographed, cell viability was determined, and cell growth curves were drawn.

[0063] 1.3 Determination of cell viability

[0064] Cell viability was determined using methylthiazolium tetrazolium (MTT). 10 μL of 5 mg / mL MTT solution was added to each well and incubated for 4 hours. The cell supernatant was removed and 150 μL of DMSO was added to each well. Then, after shaking at room temperature for 10 minutes, the absorbance of each well was measured at a wavelength of 490 nm and a reference wavelength of 630 nm using an ELx 800 microplate reader (Bio-Tek Instruments Inc., Winooski, VT, USA). Cell viability was calculated using the following formula: Cell viability (%) = (OD value of experimental group) 490 - Experimental group OD 630 ) / (control group OD 490 -Control group OD 630 ) × 100. Each treatment group had 6 replicates.

[0065] 1.4 Effects of different hydrogen peroxide concentrations and exposure time on CEHs cell viability

[0066] Primary chicken embryo hepatocytes were cultured at 1.0×10 5 Cells were inoculated into 96-well plates with 50% CO2 / mL. After the cells reached about 80% confluency in a 37°C cell culture incubator with 5% CO2, different concentrations of H2O2 (0, 0.25, 0.5, 1.0, 2.0, and 4.0 mM) were diluted with cell culture medium without serum and antibiotics, and cell viability was detected at 6, 12, and 24 h, respectively.

[0067] 1.5 Effects of different CPE concentrations and exposure time on CEHs cell viability

[0068] The cells were cultured at 1.0 × 10 5 Cells were inoculated into 96-well plates at 400 μg / mL and treated with different concentrations of CPE (0, 5, 10, 20, and 40 μM) diluted in cell culture medium without serum and antibiotics. Cell viability was detected at 6, 12, and 24 h, respectively.

[0069] 1.6 Protective effect of CPE on cells induced by H2O2

[0070] The cells were cultured at 1.0 × 10 5 Cells were seeded with CPE at a final concentration of 0, 5, 10, 20, and 40 μM after mixing with 1 mM H2O2 for 12 h to detect cell viability.

[0071] 1.7 CPE alleviates H2O2-induced cellular oxidative stress

[0072] CEHs cells were cultured at a rate of 1.0×10 5 / mL were inoculated into 6-well plates. After reaching 80% confluence, four treatment groups (control group, 1mM H2O2 group, 10μM CPE group and 1mM H2O2+10μM 6-Gin group) were set up with 3 replicates in each treatment group. Flow cytometry detection, antioxidant enzyme activity detection and inflammatory factor detection were performed after 12 hours of treatment.

[0073] 1.8 Flow cytometry apoptosis assay

[0074] Apoptotic cell death was assessed using the Annexin V-FITC / PI Cell Apoptosis Detection Kit (#G1511 (ServicebioBiotechnology, Wuhan, China)). Cells were trypsinized with EDTA-free trypsin, and the culture medium was removed by centrifugation at 1,000 rpm for 5 minutes. The cells were then washed twice with 4°C pre-cooled PBS. Binding Buffer (10×) was diluted to a 1× working solution of Binding Buffer (1 mL of 10× Binding Buffer was added to 9 mL of sterile deionized water). 100 μL of 1× working solution of Binding Buffer was added to the cell pellet and the cells were resuspended. 5 μL of Annexin V-FITC was added, followed by 5 μL of Propidium Iodide and mixed thoroughly. The cells were incubated at room temperature in the dark for 15 minutes. After staining and incubation, 400 μL of 1× working solution of Binding Buffer was added to each tube, mixed thoroughly, and analyzed using a CytoFLEX flow cytometer (Beckman, Brescia, CA, USA). Annexin V-FITC green fluorescence was detected in the FITC channel (FL1), and PI red fluorescence was detected in the PI channel (FL2). Flow cytometer parameters were as follows: excitation wavelength Ex = 488 nm, emission wavelengths FL1 (Em = 525 ± 20 nm); FL2 (Em = 585 ± 21 nm). FlowJo software was used to analyze the proportion and number of cells in different states.

[0075] 1.9 Data Statistics and Analysis

[0076] Data were presented as mean ± standard deviation (SD) and one-way analysis of variance (ANOVA) was performed using SPSS 23.0 software (IBM Corporation, Armonk, NY, USA). All images were generated using GraphPad Prism 9 (GraphPad Software Inc, San Diego, CA, USA). P-value < 0.05 was considered statistically significant, P < 0.01 was considered extremely significant, and P > 0.05 was considered not statistically significant.

[0077] 2 Results

[0078] 2.1 Effects of different H2O2 concentrations and time on CEHs cell viability

[0079] CEHs cells were exposed to different concentrations of H2O2 (0, 0.25, 0.5, 1, 2, and 4 mM) for 6, 12, and 24 h. As shown in Table 7, CEHs cell viability gradually decreased with increasing H2O2 concentration and treatment time. When CEHs were treated with H2O2 at a concentration of 1 mM for 12 h, the CEHs viability was 76.69%, making it suitable as a subsequent injury model.

[0080] Table 7 Effects of different concentrations of H2O2 treatment for different times on CEHs cell viability

[0081]

[0082] In the same column, significant differences at the P < 0.01 level are represented by different capital letters (A, B, and C), while significant differences at the P > 0.05 level are represented by the same capital letters. In the same row, significant differences at the P < 0.05 level are represented by different lowercase letters (a, b, candd), while non-significant differences at the P > 0.05 level are represented by the same lowercase letters.

[0083] 2.3 Effects of different CPE concentrations and exposure time on CEHs cell viability

[0084] As shown in Table 8, compared with the control group, 5, 10, and 20 μM CPE did not show significant damage to the cells (P>0.05), but at 40 μM, it showed an inhibitory trend. The cell viability reached the highest level (112.4%) after treatment with 10 μM CPE for 12 hours.

[0085] Table 8 Effects of different concentrations of CPE incubated for different times on cell viability

[0086]

[0087] In the same column, significant differences at the P < 0.01 level are represented by different capital letters (A, B, C), while non-significant differences at the P > 0.05 level are represented by the same capital letters. In the same row, significant differences at the P < 0.05 level are represented by different lowercase letters (a, b, candd), while non-significant differences at the P > 0.05 level are represented by the same lowercase letters.

[0088] 2.4 CPE alleviates H2O2-induced damage to CEHs cells

[0089] After incubation with 10 μM CPE and 1 mM H₂O₂ for 12 h, the cell viability was shown in Table 9. The cell viability in the 1 mM H₂O₂ treatment was 77.28%, while the cell viability increased to 101.11% after the addition of 10 μM CPE (P < 0.05). These results indicate that co-treatment with 10 μM CPE can alleviate H₂O₂-induced damage in CEHs cells.

[0090] Table 9 CPE alleviates H2O2-induced damage to CEHs cells

[0091] control group 1mMH202 10μM CPE 10μM CPE+1mM H202 Cell viability% <![CDATA[100±3.8 a ]]> <![CDATA[77.28±2.14 b ]]> <![CDATA[103.12±3.24 a ]]> <![CDATA[101.11±2.15 a ]]>

[0092] Experiment 3: Animal experiment on compound plant extract (CPE) alleviating heat stress in broiler chickens

[0093] 1 Animal testing

[0094] 1.1 Feeding experiment design and grouping

[0095] Each kg of CPE contains 300 g of rutin extract + 300 g of quercetin extract + 100 g of Prunella vulgaris extract (20:1), and the remaining carrier is anhydrous glucose.

[0096] 300 healthy AA broilers (half male and half female) with no difference in body weight were selected. After 21 days of feeding the broiler diet (Table 1), 120 broilers with the same body weight were selected and divided into 3 treatment groups, with 5 replicates in each treatment group and 8 broilers in each replicate (half male and half female). The groups were as follows:

[0097] Control group: temperature was controlled at (23±1)℃, humidity was about 60%;

[0098] Group B: heat stress group, the temperature was controlled at (33±1)℃ and the humidity was about 70%.

[0099] Group C: heat stress + CPE group (0.5 kg / ton), temperature controlled at (33±1)°C, humidity about 70%.

[0100] The experimental period was 21 days, and the dietary ratios are shown in Table 10. The animals were housed in multi-layer cages with 24-h light and free access to food and water.

[0101] Table 10 Broiler feed ratio

[0102]

[0103]

[0104] Note: The diet was formulated in accordance with the NRC (1994) broiler feeding standards. Premix includes (per kg of complete feed): VA 12000 IU; VD 33000 IU; VE 20 IU; VK 31.0 mg; VB 12.0 mg; VB 63.5 mg; VB 120.01mg; Copper (Cu) (ascopper sulfate) 8mg; Iron (Fe) (asferrous sulfate) 100mg; Manganese (Mn) (asmanganese sulfate) 80mg; Zinc (Zn) (aszinc oxide) 60mg; Iodine (I) (ascalcium iodate) 0.45mg; Selenium (Se) (assodium selenite) 0.35mg; Biotin 0.15mg; Folic acid 1.25mg; Riboflavin (VB2, riboflavin) 6mg; Nicotinic acid 35mg; Calcium pantothenate 10mg. Crude protein, calcium, and total phosphorus contents are measured values; the rest are calculated values.

[0105] 2. Measurement indicators and methods

[0106] 2.1 Production performance measurement

[0107] Record daily feed intake and recover spilled feed; observe and record the health status, mortality, and diarrhea of ​​broiler chickens. Weigh the chickens after fasting for 12 hours on day 21 and day 42, and calculate the average daily gain (ADG), average daily feed intake (ADF), feed conversion rate (F / G), and mortality rate of each replicate. Mortality rate = number of deaths / total number of chickens.

[0108] 2.2 Metabolic tests and related index determination

[0109] A complete manure collection method was used. Manure pans covered with black garbage bags were placed under the chicken coops to collect feces from day 37 to 39. Debris, such as dander, feathers, and feed, was promptly removed. Daily manure was mixed, and 10% sulfuric acid was added dropwise to fix nitrogen. The mixture was then frozen in a -20°C refrigerator. After the experiment, the manure collected on day 3 was mixed, weighed, and sampled. The total manure weight and sample weight were recorded. The mixture was then dried at 60-65°C, allowed to fully recover naturally for 24 hours, weighed, and recorded. The mixture was then crushed and passed through a 40-mesh sieve for later use.

[0110] Crude protein in feed and fecal samples was determined according to the national standard GB / T6432-94; crude fat was determined according to the national standard GB / T6433-2006; calcium was determined by complexometric titration with disodium EDTA; and phosphorus was determined according to the national standard GB / T6437-2002. The two toxins in feed and samples were determined according to the Baifa kit procedures.

[0111] 2.3 Serum antioxidant enzyme assay

[0112] From each replicate, blood was collected from the carotid artery of a chicken of near-average weight. 5 mL of blood was collected from each chicken and centrifuged at 3000 rpm for 10 minutes to obtain serum. Serum levels of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-PX), total antioxidant capacity (T-AOC), and malondialdehyde (MDA) were determined using kits produced by the Nanjing Jiancheng Bioengineering Research Institute.

[0113] 3 Test results

[0114] 3.1 Growth performance

[0115] During the 22-42 days of the experiment, the average feed intake and daily weight gain of the heat stress group were significantly lower than those of the other two groups, and the feed-to-meat ratio was significantly higher than that of the other two groups (P<0.05). After adding CPE, the above indicators returned to no significant difference with the control group (P>0.05).

[0116] Table 11 Effects of CPE on heat stress performance of broiler chickens

[0117] control group Heat stress group Heat stress + CPE group Average feed intake g <![CDATA[121.35±8.79 a ]]> <![CDATA[89.35±7.21 b ]]> <![CDATA[116.25±8.32 a ]]> Average daily weight gain (g) <![CDATA[59.27±7.31 a ]]> <![CDATA[36.89±4.21 b ]]> <![CDATA[58.21±7.32 a ]]> Feed-to-meat ratio <![CDATA[2.04±0.12 ab ]]> <![CDATA[2.42±0.27 a ]]> <![CDATA[1.99±0.32 b ]]> mortality rate% 0 30 5

[0118] 3.2 Nutrient metabolism rate

[0119] During the 22-42 days of the experiment, the average feed intake and daily weight gain of the heat stress group were significantly lower than those of the other two groups, and the feed-to-meat ratio was significantly higher than that of the other two groups (P < 0.05). After adding CPE, the above indicators returned to no significant difference with the control group (P > 0.05).

[0120] Table 12 Effect of CPE on nutrient metabolism rate of broiler chickens under heat stress (%, n=4)

[0121] control group Heat stress group Heat stress + CPE group crude protein <![CDATA[62.31±1.35 a ]]> <![CDATA[49.34±2.11 b ]]> <![CDATA[54.61±3.04 a ]]> crude fat <![CDATA[72.38±2.32 a ]]> <![CDATA[59.23±1.11 b ]]> <![CDATA[71.19±3.21 a ]]> Ca <![CDATA[41.29±0.89 a ]]> <![CDATA[29.34±1.08 b ]]> <![CDATA[38.35±1.31 a ]]> p <![CDATA[49.27±2.13 a ]]> <![CDATA[27.12±0.64 b ]]> <![CDATA[47.29±1.31 a ]]>

[0122] 3.3 Serum antioxidant enzyme activity

[0123] During the 22-42 days of the experiment, the antioxidant enzymes in the heat stress group were significantly lower than those in the other two groups, and the MDA content was significantly higher than those in the other two groups (P < 0.05). After adding CPE, the above indicators returned to the level of no significant difference with the control group (P > 0.05).

[0124] Table 13 Effects of CPE on the activity of antioxidant enzymes in serum of broiler chickens subjected to heat stress (n=4)

[0125] control group Heat stress group Heat stress + CPE group Total antioxidant capacity (mmol / mL) <![CDATA[0.61±0.02 a ]]> <![CDATA[0.31±0.02 b ]]> <![CDATA[0.59±0.02 a ]]> Catalase (u / mL) <![CDATA[8.89±0.29 a ]]> <![CDATA[4.39±0.23 b ]]> 8.87±0.69a SOD (u / mL) <![CDATA[42.08±4.95 b ]]> <![CDATA[10.98±0.38 c ]]> <![CDATA[51.49±0.27 a ]]> GSH-PX (μmol / mL) <![CDATA[503.12±12.12 b ]]> <![CDATA[283.47±18.21 b ]]> <![CDATA[499.31±14.28 a ]]> MDA (nmol / mL) <![CDATA[2.88±0.13 b ]]> <![CDATA[4.15±0.12 a ]]> <![CDATA[2.99±0.13 b ]]>

Claims

1. A composite plant extract for alleviating heat stress in poultry, characterized in that: The invention is prepared by mixing quercetin, rutin and selfheal extract in a mass ratio of 3:3:1, wherein the purity of quercetin is 98% and the purity of rutin is 95%. The selfheal extract is prepared by the following method: selfheal herbs are decocted with water 1-3 times, each time for 2-3 hours, filtered, the decoction is concentrated, 70-90% ethanol is added to adjust the alcohol content to 55-60%, the supernatant is collected and concentrated to obtain the extract.

2. Use of a composite plant extract for alleviating heat stress in poultry in the preparation of a functional feed additive for alleviating heat stress in poultry, characterized in that: 300 g of rutin extract, 300 g of quercetin extract and 100 g of selfheal extract, with the remainder being anhydrous glucose, are prepared into 1 kg of a composite plant extract. The quercetin, rutin and selfheal extract are mixed in a mass ratio of 3:3:

1. The purity of quercetin is 98%, and the purity of rutin is 95%. The selfheal extract is prepared by the following method: selfheal herbs are decocted with water 1-3 times for 2-3 hours each time, filtered, and the decoction is concentrated. 70-90% ethanol is added to make the alcohol content 55-60%, the supernatant is collected and concentrated to obtain an extract.

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