Aleppo avens n-acetone extract and application thereof in treating necrotic enteritis

Abstract

The invention relates to an aleppo avens n-acetone extract and application thereof in treating necrotic enteritis. The aleppo avens n-acetone extract is obtained through a reflux extraction method, and the extraction conditions are as follows: the acetone concentration is 50% or 75%, the reaction time is 0.5 h or 1 h, the material-liquid ratio is 1: 5 or 1: 10, and the extraction temperature is 65 DEG C. According to the invention, a reflux extraction method is adopted to extract the aleppo avens, and the obtained aleppo avens n-acetone extract contains various compounds such as ellagic acid, buddleoside, 4, 4 '-dimethyl ellagic acid and the like; the aleppo avens n-acetone extract can increase the villus height of duodenum and jejunum and the ratio of villus height to crypt depth, maintain the morphological structure in the duodenum intestinal tract, increase the content of acetic acid, propionic acid and butyric acid in cecum, and significantly reduce necrotic enteritis lesion.

Description

Technical Field

[0001] The invention relates to a blue cloth n-acetone extract and application thereof in treating necrotic enteritis. Background Art

[0002] Necrotic enteritis is an intestinal bacterial disease caused by Clostridium perfringens, often accompanied by symptoms such as diarrhea, and has the characteristics of high morbidity and mortality, which greatly damages human health and undermines the healthy development of the poultry industry.

[0003] At present, antibiotics such as penicillin, tylosin, lincomycin, and amoxicillin are mostly used to treat necrotic enteritis in chickens. However, due to the abuse and long-term irrational use of antibiotics in the treatment of necrotic enteritis in chickens, the drug resistance rate has gradually increased and the risk of drug residues has increased. The abuse, misuse, and irrational use of feed antibiotics have seriously damaged the health of animals and humans. The most studied alternative antibiotic products are mainly probiotics, plant extracts, etc.

[0004] Chinese herbal medicine ingredients are natural compounds with low toxicity, no residue, no pollution, etc. The biologically active substances in Chinese herbal medicine extracts, such as flavonoids, tannins (polyphenols), terpenes, and essential oils, have anti-inflammatory, antibacterial, and antioxidant pharmacological activities. In recent years, studies have found that many Chinese herbal medicine extracts and Chinese herbal medicine preparations have the effect of treating necrotic enteritis in chickens. Chinese herbal medicine is of great significance for the development of natural and efficient new drugs for the treatment of animal diseases.

[0005] GeiHerba is the dried whole herb of Geumjaponicum Thunb.var.chinense F.Bolle, a plant of the genus Geum in the family Rosaceae. It is also known as water bayberry, wind-chasing seven, and roadside green. It is included in the 2020 edition of the "Chinese Pharmacopoeia" and is widely distributed in my country. The whole herb can be used as medicine, and has the effects of invigorating qi and blood, removing phlegm and relieving asthma, promoting blood circulation and detoxifying, replenishing deficiency and benefiting the kidney, and removing wind and relieving pain. GeiHerba is rich in chemical components, and the main components include triterpenes, flavonoids, tannins, lignans, volatile oils and other compounds. It has anti-inflammatory, antihypertensive, anticoagulant, hypoglycemic, anti-tumor, antioxidant, antibacterial, and anti-diarrhea pharmacological effects. How to improve the effective substances of the extract of GeiHerba is an important research direction. Summary of the invention

[0006] In order to further explore the pharmacological mechanism of anti-inflammatory and anti-diarrhea of ​​Lanbuzheng, the present invention first takes Lanbuzheng as the research object, analyzes the chemical components in the Lanbuzheng acetone extract; explores the total tannin content in the Lanbuzheng acetone extract, and optimizes the process flow to obtain the optimal parameters for the extraction of total tannins from Lanbuzheng; evaluates the in vitro anti-inflammatory effect of the Lanbuzheng acetone extract based on the lipopolysaccharide (LPS)-induced mouse small intestinal ModeK cell inflammation model; determines the antibacterial effect of the Lanbuzheng acetone extract by the in vitro antibacterial test of the Lanbuzheng acetone extract on Clostridium perfringens, and studies the effect of the Lanbuzheng acetone extract on preventing and treating poultry necrotic enteritis by establishing an artificial model of necrotic enteritis with chickens as the model. The present invention provides a research direction for the anti-inflammatory and anti-diarrhea mechanism of action of Lanbuzheng, and provides a theoretical basis for the clinical application of Lanbuzheng and the development of related new drugs.

[0007] The main research contents and results are as follows:

[0008] 1. Analysis of chemical components of acetone extract of blue cloth: The total tannins of blue cloth were extracted by heating reflux method with a solid-liquid ratio of 1:10, 70% acetone solvent, extraction temperature of 65°C, and extraction time of 1h. The components in the acetone extract of blue cloth were analyzed and studied by HPLC-ESI-Q-TOF-MS technology. More than 29 compounds were obtained, among which ellagic acid had the highest content and 23-hydroxypotentillac acid had the lowest content.

[0009] 2. Optimization of the acetone extraction process of total tannins from indigofera: (1) A method for determining the content of total tannins from indigofera was established using UV-visible spectrophotometry. Using gallic acid as the standard, it was found that the determination of total tannins from indigofera using the OD value at 760nm had good correlation, accuracy and repeatability; (2) Based on three single-factor experiments on solid-liquid ratio, acetone concentration and extraction time, the optimal process conditions for the acetone extraction of total tannins from indigofera were determined through orthogonal design experiments. The results showed that the optimal process parameters for indigofera were: reflux extraction method, acetone concentration of 50%, reaction time of 0.5h, solid-liquid ratio of 1:5, and extraction temperature of 65°C. Under the extraction process conditions, the total tannin content of the extract can reach 30.2840mg / g.

[0010] 3. Animal experiment on the prevention and treatment of necrotic enteritis by acetone extract of blue cloth. An artificial model of necrotic enteritis in chickens was established to observe intestinal lesions and pathological histological characteristics of different intestinal segments (duodenum, jejunum, ileum). GC, high-throughput sequencing, qPCR and other methods were used to detect cecal short-chain fatty acids, intestinal flora, jejunal mucosal mRNA, etc. The results showed:

[0011] (1) The intestinal lesions were scored visually according to the scoring standard for small intestinal lesions in chickens with necrotic enteritis. The results showed that compared with the disease control group, the medium-dose Lanbu Zheng prevention group was significantly reduced (P<0.05), while there was no significant difference between the low-dose and high-dose Lanbu Zheng prevention groups and the pomegranate peel control group.

[0012] (2) The jejunum, ileum and duodenum of each group were sliced ​​and observed under a microscope for intestinal villus length and crypt depth. The values ​​were measured and the ratio of villus length to crypt depth was calculated to evaluate the improvement effect of Lanbuzheng extract on intestinal tissue morphology. The results showed that the villus length of the jejunum and duodenum in the high-dose Lanbuzheng prevention group was significantly higher than that in the disease control group (P), and the villus length / crypt depth was significantly higher than that in the disease control group (P<0.05); compared with the disease control group, there was no significant difference in villus length in the low- and medium-dose Lanbuzheng prevention groups, and there was no significant difference in crypt depth among the drug prevention groups.

[0013] (3) The genomic DNA of the small intestinal contents was extracted, and the number of Clostridium perfringens was detected by qPCR. The results showed that the content of Clostridium perfringens in the small intestinal contents of the low-, medium-, and high-dose Lanbuzheng prevention groups was significantly lower than that in the disease control group (P < 0.05).

[0014] (4) DNA from the small intestine was extracted for 16s rRNA gene high-throughput sequencing, and the intestinal flora was analyzed. The results showed that compared with the diseased group, the Sobs index of the blue cloth medium-dose prevention group was significantly increased (P < 0.05), indicating that the blue cloth acetone extract can improve the richness of intestinal microorganisms; compared with the diseased group, there was no significant difference in the Shannon index of microbial community diversity in each prevention group. The results of species abundance analysis at the phylum level showed that the relative abundance of Firmicutes in the blank control group (O group) was significantly lower than that in other groups (P < 0.05); the relative abundance of Actinobacteria in the blank control group was significantly higher than that in other groups (P < 0.05). The results of species abundance analysis at the genus level showed that compared with the fish meal control group, the relative abundance of Lactobacillus in the disease control group was significantly increased, and the relative abundance of Clostridium sensu lato_1 was significantly decreased (P < 0.05); compared with the disease control group, the relative abundance of Lactobacillus in the Lanbuzheng prevention groups of each dose was significantly decreased (P < 0.05), the relative abundance of Rombutsella was significantly increased (P < 0.05), the relative abundance of Filamentous Bacteria in the Lanbuzheng medium-dose prevention group was significantly increased (P < 0.05), the relative abundance of Enterococcus in the Lanbuzheng low-dose prevention group was significantly increased (P < 0.05), and the relative abundance of Clostridium in the Lanbuzheng high-dose prevention group was significantly increased (P < 0.05).

[0015] (5) RNA was extracted from the jejunal mucosa, and transcriptome sequencing was performed to screen the differentially expressed genes (DEGs) in different treatment groups, and enrichment analysis was performed. The results of GO functional enrichment analysis showed that the differentially expressed genes between the disease control group and the fish meal control group were mainly enriched in nuclear chromosome separation, cell cycle, cell division, cell mitosis cycle and other processes in terms of biological processes; in terms of cellular components, they were mainly enriched in chromosome centromere region, chromosome region, chromosome organization and other components; in terms of molecular functions, they were mainly enriched in functions such as cell cycle regulation. The DEGs between the blue cloth positive group and the disease control group were mainly enriched in organic acid metabolism, small molecule metabolism, lipid metabolism, carboxylic acid metabolism, cell cycle, chromosome separation, etc.; in terms of molecular functions, they were mainly enriched in redox-driven active transmembrane transporter activity, primary active transmembrane transporter activity, electron transfer activity, etc.

[0016] The results of KEGG functional enrichment analysis showed that the pathways with significant enrichment of differentially expressed genes between the disease control group and the fish meal control group mainly included the FoxO transcription factor signaling pathway, etc.; the pathways with significant enrichment of DEGs between the blue cloth group and the disease control group mainly included chemical carcinogenesis-reactive oxygen, oxidative phosphorylation, chemokine signaling pathway, galactose metabolism, FoxO transcription factor signaling pathway, etc.

[0017] (6) The contents of the cecum were extracted and the content of short-chain fatty acids was detected by gas chromatography. The results showed that compared with the disease control group, the contents of acetic acid, propionic acid, and butyric acid in the Lanbu Zhengzhong and high-dose prevention groups were significantly increased (P<0.05).

[0018] in conclusion:

[0019] 1. The present invention uses an ultra-high performance liquid chromatography-time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) analysis method to analyze the components of the acetone extract of Indigofera chinensis, and obtains 29 suspected compounds, of which 23 can be inferred to have structural formulas. In descending order, they are tannins and flavonoids such as ellagic acid, buddleja glycoside, and 4,4'-dimethylellagic acid, providing an experimental basis for the subsequent optimization of the acetone reflux extraction process of Indigofera chinensis.

[0020] 2. Through orthogonal design experiments, the optimal extraction process of total tannins from blue cloth was determined as follows: reflux extraction, extraction solvent acetone concentration 50%, extraction time 0.5h, solid-liquid ratio 1:5, extraction temperature 65°C. The total tannin content obtained can reach 30.2840mg / g.

[0021] 3. The results of the artificial model test of necrotic enteritis in chickens showed that the acetone extract of blue cloth can significantly reduce the lesions of enteritis, specifically in the following aspects:

[0022] (1) Lanbuzheng can significantly reduce the lesion score of necrotic enteritis; Lanbuzheng has a significant improvement effect on the intestinal morphology of the jejunum and duodenum, and the height of intestinal villi and its ratio to crypt depth are significantly increased.

[0023] (2) Lanbuzheng can significantly reduce the number of netB-positive Clostridium perfringens in the small intestine; Lanbuzheng can significantly increase the relative abundance of beneficial bacteria such as Rombutsella, Segmented filamentous bacteria and Clostridium, and has different degrees of improvement on the small intestinal flora disorder caused by necrotic enteritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the total ion current of LC-MS of acetone extract of blue cloth under -ESI;

[0025] Figure 2 This is the total ion current of LC-MS of acetone extract of blue cloth under +ESI;

[0026] Figure 3 This is the full wavelength scanning spectrum of gallic acid;

[0027] Figure 4 This is the full wavelength scanning spectrum of the acetone extract of blue cloth;

[0028] Figure 5 This is the standard curve of gallic acid;

[0029] Figure 6 The test results for the range of extraction solvent concentrations;

[0030] Figure 7 This is the test result diagram for the range of solid-liquid ratio;

[0031] Figure 8 This is the test result diagram for the extraction time range;

[0032] Fig. 9 It is the anatomical lesion diagram;

[0033] Fig.10 It is a lesion scoring diagram;

[0034] Fig.11 This is a picture of the colony morphology of Clostridium perfringens;

[0035] Fig.12 The height of duodenal villi and crypt depth and the ratio of the two are shown in each group;

[0036] Fig.13 The height of jejunal villi and crypt depth and the ratio of the two are shown in each group;

[0037] Fig.14 Figure 3 shows the height of ileal villi, crypt depth and their ratio in each group. DETAILED DESCRIPTION

[0038] Analysis of Chemical Components of Acetone Extract from Blue Cloth

[0039] Experimental Materials

[0040] Lan Buzheng: Purchased from Dasanlin Pharmaceutical Group Co., Ltd., identified by Associate Professor Cheng Xuanxuan of the School of Chinese Medicine of Guangdong Pharmaceutical University as the dried whole herb of Geum aleppicum Jacq. of the Rosaceae family.

[0041] Experimental methods

[0042] Preparation of test solution

[0043] Accurately weigh 1.0g of blue cloth powder and pour it into a round-bottom flask, add 10 times the amount of 70% acetone, reflux in a 65°C water bath for 0.5h, and after returning to room temperature, filter, discard the initial filtrate, take the subsequent filtrate, and evaporate the solvent to obtain the product.

[0044] Preparation of reference solution

[0045] Accurately weigh appropriate amounts of gallic acid, pomegranate peel tannin, catechin, proanthocyanidin B2, corilagin, ellagic acid, and kaempferol reference substances into a 5 mL volumetric flask, dilute to volume with distilled water, and shake well to obtain a reference substance solution with a concentration of 1 μg / mL.

[0046] Analysis conditions

[0047] (1) Chromatographic conditions

[0048] Chromatographic column: ACQUITY UPLC BEH C18, (2.1×100mm, 1.7pm) Mobile phase: acetonitrile: water (both containing 0.1% formic acid)

[0049] (2) Mass spectrometry conditions: Ionization mode: positive and negative ion scanning mode; capillary voltage: 2.5 kV; cone voltage: 35 V; source temperature: 120 °C; curtain gas: 40 L / h; nebulizer: 6.5 Bar; desolvation gas temperature: 350 °C; desolvation gas: 800 L / h.

[0050] Experimental Results

[0051] according to Figure 1The relative molecular mass of the compound was obtained from the main ion peaks shown above, and the elemental components were fitted by mass spectrometry analysis software. The molecular formula and relative molecular mass of the suspected compound were input into Sci-finder and the Chinese Natural Product Chemical Component Database to search for chemical components related to Lanbuzheng. The structure of the compound was inferred based on the compound ion fragments in the primary and secondary mass spectra, and the compound structure was further determined. The suspected compound components were compared with the previous analysis results of Lanbuzheng components to obtain the most likely suspected compound, and the compounds in the Lanbuzheng acetone extract were preliminarily inferred. The experimental results are shown in Figure 1 and Figure 2 , 29 suspected compounds were obtained from the acetone extract of blue cloth, of which 23 were deduced to have certain compound formulas. The order of content was ellagic acid, budding glycoside, 4,4'-dimethyl ellagic acid, (4R)-2ɑ,3β,19ɑ-trihydroxyursu-12-ene-23,28-diacid, 2ɑ,3ɑ,19ɑ,23-tetrahydroxyursu-12-ene-28-acid, 3,6,19,23-tetrahydroxy-12-ursula-28-acid, 3,3'-dimethyl ellagic acid, epicatechin, cannabinoids, 3-methyl ellagic acid, Aromatic acid, 9,12,13-trihydroxy-10-octadecenoic acid, astragaloside, gallic acid, witch hazel tannin, methyl 1-ethoxy-4,5-dioxo-3-pyrrolidinecarboxylate, kaempferol, 2,3,23-trihydroxy-12-oleanole-28-acid, 1-hydroxy-2-oxo-oleanolic acid, [(8e,11z)-1-carboxyheptane-8,11-dien-7-yl]oxyacyl, linoleic acid, potentilla acid, 3,3'-dimethylellagic acid-4-O-β-D-glucoside, 23-hydroxypotentilla acid.

[0052] According to the ingredients, they can be classified into:

[0053] (1) Terpenoids: Potentilla acid, 23-hydroxypotentilla acid, 3,6,19,23-tetrahydroxy-12-ursolic acid, 2,3,23-trihydroxy-12-oleanole-28-acid, 1-hydroxy-2-oxocarpic acid; (2) Flavonoids: budding glycoside, kaempferol, epicatechin; (3) Tannins: ellagic acid, 4,4'-dimethylellagic acid, 3,3'-dimethylellagic acid, cannabinol, 3-methylellagic acid, gallic acid, 3,3'-dimethylellagic acid-4-O-β-D-glucoside, witch hazel tannin; (4) Volatile oil compounds: linoleic acid ; (5) Acid compounds: (4R)-2ɑ,3β,19ɑ-trihydroxyursu-12-ene-23,28-diacid, 2ɑ,3ɑ,19ɑ,23-tetrahydroxyursu-12-ene-28-acid, 9,12,13-trihydroxy-10-octadecenoic acid; (6) Ester compounds: methyl 1-ethoxy-4,5-dioxo-3-pyrrolidinecarboxylate; (7) Acyl compounds: [(8e,11z)-1-carboxyheptane-8,11-dien-7-yl]oxyacyl; There were 6 peaks with higher responses in the mass spectrum that could not be identified, which were confirmed by infrared spectroscopy analysis and nuclear magnetic resonance spectrum analysis.

[0054] In this experiment, the composition of the acetone extract of Lanbu Zheng was analyzed by ultra-high performance liquid chromatography-time of flight mass spectrometry (UHPLC-Q-TOF-MS) analysis method, and 29 suspected compounds were obtained, of which 23 were deduced to have certain compound structures. The order of content was ellagic acid, buddleja glycoside, 4,4'-dimethylellagic acid, etc., which provided an experimental basis for the subsequent optimization of the acetone extraction process of Lanbu Zheng.

[0055] Study on the Extraction Technology of Total Tannin from Blue Cloth

[0056] Lan Buzheng: Purchased from Dasanlin Pharmaceutical Group Co., Ltd., identified by Associate Professor Cheng Xuanxuan of the School of Chinese Medicine of Guangdong Pharmaceutical University as the whole herb of Geum aleppicum Jacq. of the Rosaceae family.

[0057] Reflux Extraction Technology of Total Tannins from Blue Cloth

[0058] Accurately weigh 1.0 g of blue button powder, add it into a round-bottom flask, add 100 mL of 75% acetone solution, heat it in a water bath and reflux for 0.5 h. After it returns to room temperature, filter it and discard the initial filtrate (10-20 mL). The subsequent filtrate obtained is the extract.

[0059] Determination of total tannin content

[0060] (1) Preparation of test solution: Accurately measure 20 mL of the filtrate into a 100 mL brown volumetric flask, dilute to the mark with water, and shake well to prepare the test solution.

[0061] (2) Preparation of reference solution: Accurately weigh 50 mg of gallic acid reference substance, place it in a 100 mL brown volumetric flask, add water to dissolve and dilute to the mark, shake well. Accurately measure 5 mL again, place it in a 50 mL brown volumetric flask, dilute to the mark with water, shake well. This means that each 1 mL contains 0.05 mg of gallic acid.

[0062] (3) Selection of measurement wavelength: Accurately measure 2 mL of gallic acid reference solution and place it in a 25 mL brown volumetric flask, add 1 mL of forlin phenol test solution, then add 10 mL of water, dilute to the scale with 29% sodium carbonate solution, shake well, and place in a dark place for 30 min. Use the corresponding reagent as blank and scan in the wavelength range of 400 to 760 nm according to the UV-visible spectrophotometry method to determine the wavelength with maximum absorbance, and use this as the detection wavelength of gallic acid.

[0063] (4) Preparation of standard curve: Accurately measure 0 mL, 0.2 mL, 0.5 mL, 1 mL, 2 mL, 3 mL, and 4 mL of the reference solution and place them in 25 mL brown volumetric flasks, respectively. Add 1 mL of forlin phenol test solution to each flask, and then add 12 mL, 11.8 mL, 11.5 mL, 11 mL, 10 mL, 9 mL, and 8 mL of water, respectively. Dilute to the scale with 29% sodium carbonate solution, shake well, and place in a dark place for 30 min. Use the corresponding reagent as blank and measure the absorbance at the detection wavelength according to the UV-visible spectrophotometry. Draw a standard curve with absorbance as the ordinate and concentration as the abscissa.

[0064] (5) Measurement method

[0065] Total phenol: Accurately measure 2 mL of the test solution in a 25 mL brown volumetric flask, add 1 mL of folin phenol test solution, then add 10 mL of water, dilute to the scale with 29% sodium carbonate solution, shake well, and place in a dark place for 30 min. Use the corresponding reagent as blank and determine the absorbance according to the UV-visible spectrophotometry. Read the amount of gallic acid in the test solution from the standard curve and calculate to obtain the total phenol.

[0066] Non-adsorbed polyphenols: Accurately measure 25 mL of the test solution, add it to a 100 mL stoppered conical flask containing 0.6 g of casein, seal it, and keep it warm in a 30°C constant temperature water bath for 1 hour. Shake it from time to time, take it out, cool it, shake it well, filter it, discard the initial filtrate, accurately measure 2 mL of the subsequent filtrate, put it in a 25 mL brown volumetric flask, add 1 mL of folin phenol test solution, then add 10 mL of water, dilute it to the scale with 29% sodium carbonate solution, shake it well, and place it away from light for 30 minutes. Use the corresponding reagent as blank, and determine the absorbance according to the UV-visible spectrophotometry method. Read the amount of gallic acid in the test solution from the standard curve and calculate it.

[0067] (6) Calculation: Calculate the total phenol content and the non-adsorbed polyphenol content according to the standard curve method. The difference between the two is the tannin content. That is, tannin content = total phenol content - non-adsorbed polyphenol content

[0068] Note: During the determination, a casein adsorption blank experiment was carried out simultaneously and the blank value was calculated and subtracted.

[0069] Methodological investigation

[0070] 1. Precision test: Accurately measure 2.0 mL of gallic acid reference solution, place it in a 25 mL brown volumetric flask, add 1 mL of folin phenol test solution, then add 10 mL of water, dilute to the scale with 29% sodium carbonate solution, shake well, and place in a dark place for 30 minutes. Use the corresponding reagent as blank and determine the absorbance at the detection wavelength according to the UV-Vis spectrophotometry. Measure 6 times in succession. The precision value is estimated by its relative standard deviation RSD.

[0071] 2. Stability test: Accurately measure 2.0 mL of the test solution and place it in a 25 mL brown volumetric flask. Add 1 mL of folin phenol test solution and 10 mL of water. Dilute to the scale with 29% sodium carbonate solution, shake well, and place in a dark place for 30 min. Use the corresponding reagent as blank and determine the absorbance at the detection wavelength according to the UV-Vis spectrophotometry. Measure the absorbance of the sample at 15, 30, 45, 60, 90, 120, 240, and 360 min respectively. The stability is estimated by its RSD.

[0072] 3. Repeatability test: 1.0 g of Lanbuzheng powder was accurately weighed, for a total of 6 portions, and the test solution method was used. The total tannin content was determined according to the determination method. The repeatability was estimated by its RSD.

[0073] 4. Spiked recovery test: Accurately weigh 2.0 g of blue button powder with known content, a total of 9 portions, add gallic acid reference substance at 80%, 100%, and 120% of the sample content respectively, operate according to the test sample solution method, and determine it according to the above-mentioned total tannin content determination method. The recovery rate is estimated by its RSD.

[0074] Screening of extraction methods

[0075] 1. Reflux extraction method: Accurately weigh 1.0 g of blue button powder, add it into a round-bottom flask, add 10 mL of 75% acetone solution, reflux in a water bath for 0.5 h, after returning to room temperature, filter, discard the initial filtrate, take the subsequent filtrate, evaporate the solvent, and measure and calculate the tannin content.

[0076] 2. Cold soaking method: Accurately weigh 1.0 g of blue button powder into a 100 mL stoppered conical flask, add 10 mL of 75% acetone solution, leave at room temperature for 24 hours, filter, discard the initial filtrate, take the subsequent filtrate, evaporate the solvent, measure and calculate the tannin content.

[0077] 3. Ultrasonic extraction method: accurately weigh 1.0 g of blue button powder into a 100 mL stoppered conical flask, soak it with 10 mL of 75% acetone solution. Ultrasonic treatment for 0.5 h, filter, discard the initial filtrate, take the subsequent filtrate, evaporate the solvent, and measure and calculate the tannin content.

[0078] Screening of extraction solvents

[0079] 1. Ethanol: Accurately weigh 1.0 g of blue button powder, add it into a round-bottom flask, add 10 mL of 75% ethanol solution, reflux in a water bath for 0.5 h, after returning to room temperature, filter, discard the initial filtrate, take the subsequent filtrate, evaporate the solvent, and measure and calculate the tannin content.

[0080] 2. Acetone: Accurately weigh 1.0 g of blue bubble powder, add it into a round-bottom flask, add 10 mL of 75% acetone solution, reflux in a water bath for 0.5 h, after returning to room temperature, filter, discard the initial filtrate, take the subsequent filtrate, evaporate the solvent, measure and calculate the tannin content.

[0081] Single factor test: After the extraction method and extraction solvent are determined in two steps, the influence range of the extraction solvent concentration, solid-liquid ratio, and extraction time needs to be preliminarily determined through single factor test.

[0082] 1. Investigation of acetone concentration

[0083] Accurately weigh 2.0 g of indigofera powder and add it into a round-bottom flask. Set the extraction temperature to 65°C, the extraction time to 0.5 h, the solid-liquid ratio to 1:5, and different mass concentrations of acetone solvent as the extraction conditions. Use the water bath reflux method to conduct a single factor experiment on the extraction rate of total tannins in indigofera with different concentrations of acetone. The designed acetone concentrations are 0%, 25%, 50%, 75% and 100%, respectively. After the obtained extract is restored to room temperature, it is filtered, the initial filtrate is discarded, the subsequent filtrate is taken, the solvent is evaporated, and the tannin content therein is measured and calculated.

[0084] 2. Investigation of material-liquid ratio

[0085] Accurately weigh 2.0 g of Lanbuzheng powder, add it into a round-bottom flask, and use extraction temperature of 65°C, extraction time of 0.5 h, acetone concentration of 25%, and different solid-liquid ratios as extraction conditions. Using water bath reflux method, a single factor experiment was conducted on the extraction rate of total tannins in Lanbuzheng with different solid-liquid ratios. The designed solid-liquid ratios were 1:5, 1:7.5, 1:10, 1:12.5 and 1:15, respectively. After the obtained extract was restored to room temperature, it was filtered, the initial filtrate was discarded, the subsequent filtrate was taken, the solvent was evaporated, and the tannin content therein was measured and calculated.

[0086] 3. Investigation of extraction time

[0087] Accurately weigh 2.0 g of indigofera lanbu powder, add it into a round-bottom flask, and use extraction temperature of 65°C, solid-liquid ratio of 1:5, acetone concentration of 25%, and different extraction times as extraction conditions. Using water bath reflux method, a single factor experiment was conducted on the extraction rate of total tannins in indigofera lanbu with different extraction times. The designed extraction times were 0.5h, 1h, 1.5h, 2h and 3h, respectively. After the obtained extract was restored to room temperature, it was filtered, the initial filtrate was discarded, the subsequent filtrate was taken, the solvent was evaporated, and the tannin content therein was measured and calculated.

[0088] Orthogonal Experimental Design

[0089] According to the results of the single factor test, the optimal extraction conditions of each single factor were determined. Under the condition of fixed extraction temperature at 65 °C, a comprehensive investigation and study was conducted on the three main influencing factors of extraction acetone concentration (A), solid-liquid ratio (B), and extraction time (C). See Table 1. Take 2.0 g of Lanbuzheng powder and prepare 9 test solutions according to Table 2 to determine the tannin content.

[0090] Table 1 Orthogonal experimental design factors and levels

[0091]

[0092] Table 2 Orthogonal experiment arrangement

[0093]

[0094] Verification test

[0095] Accurately weigh 2.0 g of blue button powder, add it into a round-bottom flask, extract according to the optimal process conditions, filter it after it returns to room temperature, discard the initial filtrate, take the subsequent filtrate, evaporate the solvent, measure and calculate the tannin content, and conduct experimental verification.

[0096] Experimental Results

[0097] Standard curve: Full wavelength scanning spectra of gallic acid reference and blue cloth acetone extract samples ( Figure 3 , Figure 4) shows that both the test and reference solutions have relatively stable peaks at around 760nm, indicating that the wavelength of 760nm is more reasonable for determining the absorbance value of total tannins in the blue cloth extract. Figure 3 , Figure 4 The standard curve of gallic acid is shown in Figure 5 The calculated regression equation is y = 0.0964x + 0.0944, and the correlation coefficient R 2 =0.9993. The results showed that the concentration of gallic acid was in the range of 1-10 μg / mL, and the absorbance and concentration showed a good linear relationship.

[0098] The above method was tested for precision, and the results showed that the RSD was 0.0964%, indicating that the precision of the instrument was good. After the stability test, the results showed that the absorbance value of the test sample within 6 hours decreased with time, so it should be measured within 30 to 60 minutes. After the repeatability test, the test results showed that the RSD was 3.9090%, indicating that the repeatability of this method was good. After the sample recovery test, the results showed that the recovery rate was 92.7408% and the RSD was 2.6631%. The above methodological investigation results all meet the requirements, indicating that the method for determining the total tannin content of blue cloth is established.

[0099] Extraction method: The results are shown in Table 3. The most complete extraction was achieved by refluxing in a water bath at 65°C for 1 hour.

[0100] Table 3 Screening results of extraction methods

[0101]

[0102] Extraction solvent: The results are shown in Table 4. 75% acetone was the most complete extraction.

[0103] Table 4 Screening results of extraction solvents

[0104]

[0105] Single factor test

[0106] Effect of acetone concentration on total tannin content: Figure 6 As shown in the figure, with the increase of the concentration of the organic solvent acetone, the extraction amount of total tannins first increases and then decreases, and reaches a peak value when the acetone concentration is 50%. Therefore, the total tannin content obtained when the acetone concentration is 50% is the highest.

[0107] Effect of solid-liquid ratio on total tannin content: Figure 7 It can be seen that with the increase of the solid-liquid ratio, the extraction amount of tannins gradually decreases. Therefore, when the solid-liquid ratio is 1:5, the total tannin content is the highest.

[0108] Effect of extraction time on total tannin content: Figure 8It can be seen that as the extraction time increases, the amount of tannin extracted increases first and then decreases. Therefore, when the extraction time is 1h, the total tannin content is the highest.

[0109] Orthogonal experiment: The 9 combinations of the 3 factors and 3 levels of the experiment and the results of each group are shown in Table 5. The data in Table 5 were processed to obtain Table 6. It can be seen from Table 6 that with the total tannin content of blue cloth as the evaluation index, we can intuitively see from the data that the order of factors affecting the total tannin content of blue cloth extraction is B>C>A, that is, the solid-liquid ratio has the greatest influence on the extraction of total tannin content of blue cloth, followed by the extraction time, and the influence of the extraction solvent concentration is smaller. The optimal process selection method for the extraction of total tannins from blue cloth is A2B1C1, that is, acetone concentration of 50%, solid-liquid ratio of 1:5, and extraction time of 0.5h.

[0110] Table 5 Results of orthogonal test for extraction of blue cloth

[0111]

[0112]

[0113] Table 6 Analysis of variance of tannin yield

[0114]

[0115] Verification test: According to the optimal process conditions, acetone concentration of 50%, extraction time of 0.5h, solid-liquid ratio of 1:5, extraction temperature of 65℃, the test verification was carried out, 2.0g of Lanbuzheng powder was extracted to obtain 18mL of Lanbuzheng extract, and after dilution 125 times, the total tannin content of Lanbuzheng was measured to be 2.6919μg / mL, that is, the total tannin extraction rate was 30.2840mg / g. It is consistent with the results of the orthogonal test.

[0116] In vivo animal experiment on the prevention and treatment of necrotic enteritis by extracts of Lanbuzheng

[0117] Experimental animals: 120 one-day-old "817" chickens were purchased from Nanhai Poultry Breeding Company, Foshan City, Guangdong Province.

[0118] Experimental animal feed: The basic diet and fish meal were purchased from the feed factory of Guangzhou Jiangfeng Industrial Co., Ltd.

[0119] Fishmeal feed: 50% basic diet + 50% fishmeal

[0120] Drug preparation:

[0121] 1. Preparation of acetone extract of indigofera: 100.0 g of indigofera was extracted under the following process conditions: acetone concentration of 75%, extraction time of 1 h, extraction temperature of 65 ° C, and solid-liquid ratio of 1:10 to obtain an extract, and the solvent was evaporated by vacuum distillation. The remaining solvent was evaporated with a watch dish, and the dry powder was collected to obtain 2.55 g of indigofera extract powder. The total tannin content of indigofera was determined to be 29.0411 mg / g.

[0122] 2. Preparation of pomegranate peel extract: 10.0 g of pomegranate peel was extracted under the following extraction process conditions: acetone concentration of 75%, extraction time of 1 h, extraction temperature of 50°C, and solid-liquid ratio of 1:14 to obtain an extract, and the solvent was evaporated by reduced pressure distillation. The remaining solvent was evaporated by a watch dish, and the dry powder was collected to obtain 1.96 g of pomegranate peel extract powder.

[0123] The Clostridium perfringens JM strain (netB gene positive) was isolated and preserved by our laboratory from the intestinal lesions of diseased chickens in a broiler farm in Guangdong.

[0124] Experimental design and treatment: (1) Animal grouping and modeling and drug administration: 120 one-day-old "817" chicks were selected and fed with a basic diet until 7 days of age. The chicks with large weight differences were weighed and eliminated, and randomly divided into 8 groups (15 in each group): blank control group (O), fish meal control group (KB), disease control group (C), ampicillin prevention group (AM), low-dose Lanbuzheng group (LL), medium-dose Lanbuzheng group (LM), high-dose Lanbuzheng group (LH), and pomegranate peel control group (SM). From 7 days to 14 days of age, all groups were fed with a basic diet. After 15 days of age, except for the blank control group (O), the other groups were fed with fish meal feed (50% basic diet + 50% fish meal). The treatment methods of each group are shown in Table 7 below.

[0125] Table 7 Animal groups and treatment methods of each group

[0126]

[0127] (2) Feeding and management

[0128] One week before the animal test, the chicken house was thoroughly cleaned and disinfected. First, the chicken cages and the ground were burned with a flame gun, and then fumigated with a mixture of formaldehyde and potassium permanganate. Regular disinfection was maintained during the test. During the entire test, good ventilation was ensured, and the relative humidity was controlled at 65%-70%. After the start of feeding, the temperature was maintained at around 32°C, and then the temperature dropped by about 2-3°C every week until it dropped to room temperature. During the test, the chickens were free to eat and drink.

[0129] (3) Preparation of bacterial solution

[0130] Resuscitate Clostridium perfringens (netB gene positive) stored in the laboratory, dip the inoculation loop into the bacterial solution and draw four lines in TSC medium, and culture anaerobically for 15 hours; pick a single colony and transfer it to cooked meat medium, and culture it anaerobically at 37℃ for 15 hours; aspirate 20ml of the bacterial solution and transfer it to 400ml FT medium, and culture it anaerobically in a 42℃ water bath for 15 hours.

[0131] Scoring of small intestinal lesions and isolation and identification of Clostridium perfringens in lesions

[0132] Chickens were collectively killed by cervical dislocation at 24 days of age. Six chickens were randomly selected from each group for autopsy and observation of lesions in the duodenum, jejunum and ileum. The lesions in each group were scored separately. The scoring criteria are shown in Table 8.

[0133] Table 8

[0134]

[0135] Use a disposable swab to dip the intestinal lesion site, streak it in TSC medium, and culture it anaerobically for 16 hours; pick a single colony and inoculate it into FT medium, and culture it anaerobically for 16 hours; perform Gram staining on the bacterial solution and observe it under an optical microscope.

[0136] Experimental Results

[0137] Results of small intestinal lesion scores and isolation and identification of Clostridium perfringens in lesions

[0138] (1) Small intestinal lesion score

[0139] Three days after the infection with Clostridium perfringens, the appearance and behavior of the chickens in the blank control group and the fish meal control group were normal, while the chickens in the disease control group were listless and had diarrhea. The small intestine segments of the chickens in each group were dissected, and the results showed that no lesions were observed in the blank control group and the fish meal control group, indicating that no accidental infection occurred in the experimental chickens during the experiment; the disease control group had obvious necrotic foci ( Fig. 9 ), the intestinal wall became thinner and more transparent, the intestinal mucosa was bleeding and there was orange-yellow mucosal shedding, and the lesions of necrotic enteritis were obvious; the lesions in the drug prevention group were improved to varying degrees.

[0140] All groups were scored according to the lesion scoring criteria ( Fig.10 ), the results showed that the lesion scores of the diseased control group were significantly higher than those of the fish meal control group (P<0.05), and the lesion scores of each drug prevention group were reduced to varying degrees, among which the scores of the ampicillin prevention group and the Lanbuzheng medium-dose prevention group were significantly lower than those of the diseased control group (P<0.05); there was no significant difference between the Lanbuzheng low-dose and high-dose prevention groups and the pomegranate peel control group and the diseased control group. This indicates that the artificial disease model of necrotic enteritis in chickens was established in this experiment.

[0141] (2) Isolation and identification of Clostridium perfringens

[0142] Dip a cotton swab from the lesion and streak it on the TSC medium. The typical colonies are black, round, and smooth. Some colonies are white or light yellow. Fig.11 .

[0143] In order to further study the preventive and therapeutic effect of acetone extract of blue cloth on necrotic enteritis in experimental chickens, the height of duodenal villi (VH) and crypt depth (CD) were measured and their ratio was calculated. Fig.12 Villus height (VH): Compared with the fish meal control group, there was no significant difference in the disease control group, but the ampicillin prevention group increased significantly (P<0.05); compared with the disease control group, the ampicillin prevention group and the high-dose Lanbuzheng prevention group increased significantly (P<0.05), but there was no significant difference in the low- and medium-dose Lanbuzheng prevention groups and the pomegranate peel control group.

[0144] Crypt depth (CD): Compared with the fish meal control group, there was no significant difference in the disease control group, and the ampicillin prevention group was significantly reduced (P<0.05); compared with the disease control group, there was no significant difference in the ampicillin prevention group, the Lanbuzheng dose groups and the pomegranate peel control group.

[0145] Villus height to crypt depth ratio (VH / CD): Compared with the fish meal control group, there was no significant difference in the disease group, but it was significantly increased in the ampicillin prevention group (P<0.05); compared with the disease control group, the ampicillin prevention group and the high-dose Lanbuzheng prevention group were significantly increased (P<0.05), but there was no significant difference in the low- and medium-dose Lanbuzheng prevention groups and the pomegranate peel control group.

[0146] The acetone extract of blue cloth can increase the height of villi and the ratio of villus height to crypt depth, maintain the morphological structure of the duodenum, and a high dose of the acetone extract of blue cloth has a significant effect on the morphological structure of the duodenum.

[0147] To further study the preventive and therapeutic effect of acetone extract of blue cloth on necrotic enteritis in experimental chickens, the villus height (VH) and crypt depth (CD) of the jejunum were measured and their ratio was calculated. Fig.13 Villus height (VH): Compared with the fish meal control group, the disease control group was significantly lower (P<0.05); compared with the disease control group, the high-dose Lanbuzheng prevention group and the pomegranate peel control group were significantly higher (P<0.05), and there was no significant difference between the ampicillin prevention group and the low- and medium-dose Lanbuzheng prevention groups.

[0148] Crypt depth (CD): Compared with the fish meal control group, the disease control group was significantly increased (P<0.05); compared with the disease control group, the ampicillin prevention group, the medium-dose Lanbuzhen prevention group and the pomegranate peel control group were significantly decreased (P<0.05), and there was no significant difference between the low-dose and high-dose Lanbuzhen prevention groups.

[0149] Villus height to crypt depth ratio (VH / CD): Compared with the fish meal control group, the disease control group was significantly lower (P<0.05); compared with the disease control group, the high-dose Lanbuzheng prevention group was significantly higher (P<0.05), and there was no significant difference between the ampicillin prevention group and the low- and medium-dose Lanbuzheng prevention groups and the pomegranate peel control group.

[0150] To further study the preventive and therapeutic effect of acetone extract of blue cloth on necrotic enteritis in experimental chickens, the villus height (VH) and crypt depth (CD) of the ileum were measured and their ratio was calculated. Fig.14 Villus height (VH): Compared with the fish meal control group, the disease control group had no significant difference; compared with the disease control group, the high-dose Lanbuzheng prevention group was significantly increased (P<0.05), the ampicillin prevention group and the pomegranate peel control group were significantly decreased (P<0.05), and there was no significant difference between the low- and medium-dose Lanbuzheng prevention groups.

[0151] Crypt depth (CD): Compared with the fish meal control group, there was no significant difference in the disease control group; compared with the disease control group, the ampicillin prevention group was significantly reduced (P<0.05), and there was no significant difference in the low, medium and high doses of Lanbuzheng prevention groups and the pomegranate peel control group.

[0152] The ratio of villus height to crypt depth (VH / CD): compared with the fish meal control group, the disease control group had no significant difference; compared with the disease control group, the other drug prevention groups had no significant difference.

[0153] The present invention found that in the jejunum, compared with the fish meal control group, the disease control group can significantly reduce VH and VH / CD values ​​(P < 0.05), and the CD value is significantly increased (P < 0.05); compared with the disease control group, the high-dose prevention group of Lanbuzheng can significantly increase VH and VH / CD values ​​(P < 0.05), and the CD values ​​of the medium and high-dose prevention groups of Lanbuzheng are significantly reduced (P < 0.05). In the duodenum, the VH and VH / CD values ​​of the disease control group are reduced compared with the fish meal control group, but there is no significant difference, and the CD value is basically unchanged; the high-dose prevention group of Lanbuzheng can increase VH and VH / CD values ​​compared with the disease control group (P < 0.05), and the VH of the high-dose prevention group of Lanbuzheng is significantly increased compared with the fish meal control group (P < 0.05); and there is no significant difference in CD value between each dose group of Lanbuzheng and the fish meal control group and the disease control group. In the ileum, compared with the fish meal control group, the VH and VH / CD values ​​of the disease control group did not change significantly, while the VH value of the high-dose Lanbuzheng prevention group increased significantly (P < 0.05) and the CD value did not change significantly. This indicates that necrotizing enteritis mainly occurs in the jejunum, and there is almost no reaction in the duodenum. The high-dose Lanbuzheng group can increase the villus height and the ratio of villus height to crypt depth in the jejunum and duodenum, enhance the digestion and absorption of substances, improve the health of intestinal tissue and intestinal integrity, and thus prevent and treat necrotizing enteritis.

Claims

1. A blue cloth acetone extract, characterized in that: The product is obtained by a reflux extraction method, and the extraction conditions are: acetone concentration of 50% or 75%, reaction time of 0.5h or 1h, solid-liquid ratio of 1:5 or 1:10, and extraction temperature of 65°C.

2. The indigo plant n-acetone extract according to claim 1, characterized in that The extraction conditions of the reflux extraction method are: acetone concentration of 50%, reaction time of 0.5 h, solid-liquid ratio of 1:5, and extraction temperature of 65°C.

3. The indigo plant n-acetone extract according to claim 2, characterized in that The total tannin content of the extract reached 30.2840 mg / g by reflux extraction.

4. A method for extracting blue cloth with acetone, characterized in that the blue cloth extract is extracted by reflux extraction, and the extraction conditions are: acetone concentration of 50% or 75%, reaction time of 0.5h or 1h, solid-liquid ratio of 1:5 or 1:10, and extraction temperature of 65°C.

5. The extraction method according to claim 4, characterized in that The conditions provided by the reflux extraction method are: acetone concentration of 50%, reaction time of 0.5h, solid-liquid ratio of 1:5, and extraction temperature of 65°C.

6. The indigo plant n-acetone extract according to claim 5, characterized in that The total tannin content of the extract reached 30.2840 mg / g by reflux extraction.

7. Application of acetone extract of blue cloth in the preparation of medicine for treating necrotic enteritis in poultry.

8. The use according to claim 7, characterized in that The acetone extract of blue cloth is obtained by reflux extraction method, and the extraction conditions are: acetone concentration of 50% or 75%, reaction time of 0.5h or 1h, solid-liquid ratio of 1:5 or 1:10, and extraction temperature of 65℃.

9. A drug, characterized in that The invention comprises the indigo plant n-acetone extract as claimed in claim 1.

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