CN110548079A - Traditional Chinese medicine composition for preventing and treating chicken hepatosis syndrome as well as preparation method and application thereof - Google Patents

Traditional Chinese medicine composition for preventing and treating chicken hepatosis syndrome as well as preparation method and application thereof Download PDF

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CN110548079A
CN110548079A CN201910697244.2A CN201910697244A CN110548079A CN 110548079 A CN110548079 A CN 110548079A CN 201910697244 A CN201910697244 A CN 201910697244A CN 110548079 A CN110548079 A CN 110548079A
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chinese medicine
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medicine composition
oral liquid
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张会梅
孙亚磊
王艳玲
李有志
徐恩民
蒋贻海
付海宁
栾明娜
贺倩倩
林扬
姚德勇
钟英杰
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Qingdao National Engineering Technology Research Center Co Ltd
QINGDAO VLAND BIOLOGICAL Co Ltd
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QINGDAO VLAND BIOLOGICAL Co Ltd
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Abstract

The invention provides a traditional Chinese medicine composition for preventing and treating chicken hepatosis syndrome, a preparation method and application thereof, and belongs to the field of veterinary medicines. The technical scheme is as follows: the traditional Chinese medicine composition comprises the following raw materials in parts by weight: main drugs: 0.1 to 10 portions of white peony root, 0.1 to 10 portions of bupleurum, 0.1 to 10 portions of immature bitter orange and 0.1 to 10 portions of liquorice. The invention has the beneficial effects that: the traditional Chinese medicine composition has wide pharmacological effects, small toxic and side effects, low pollution, low medicine residue and low drug resistance, and has a good prevention and treatment effect on chicken liver injury.

Description

Traditional Chinese medicine composition for preventing and treating chicken hepatosis syndrome as well as preparation method and application thereof
Technical Field
The invention relates to the field of veterinary drugs, and in particular relates to a traditional Chinese medicine composition for preventing and treating chicken hepatosis syndrome.
Background
Chicken liver degeneration syndrome is not a single disease, but a comprehensive disease induced by various causes, and the disease is a comprehensive disease with various symptoms and lesions characterized by liver injury. The sick chicken flocks have less mental depression, lying ground is not up or is not standing, the appetite is reduced or abolished, the whole chicken flocks have less food intake and have white, yellow or green excrement, and sometimes diarrhea; a few flocks develop respiratory symptoms. The sick chicken is anemia, cockscomb is pale, and a few chickens die every day; laying hens are difficult to go up to the peak, and a few chicken flocks fall down quickly when going up to the peak. Although the treatment is improved after a long time, the original peak is difficult to return.
When the liver is stimulated by various adverse factors for a long time, excessively and excessively, the physiological functions of the liver are affected to different degrees, and the liver cells are damaged to different degrees. In addition to having an inflammatory cell infiltrate, hepatocytes undergo significant degeneration (often granulodegeneration and steatosis, sometimes vesicular degeneration) and necrosis, which may later undergo fibrosis affecting liver function.
When the liver cells are diseased, the digestion, absorption, catabolism, anabolism, conversion of nutrients and the like of the body are affected. Liver dysfunction seriously affects the normal metabolism of livestock and poultry, the excretion of decomposition products and toxic substances, the inactivation of hormone, the defense of the body are affected, glycogen cannot be normally decomposed, the supplied energy is reduced, and each organ can be harmed correspondingly, such as jaundice, anemia, emaciation, cockscomb pallor, lassitude, anorexia, the production performance is reduced, and even death occurs. Pathological changes characterized by liver degeneration, swelling, tumor, steatosis, crisp texture, hemorrhage, white dead spots, yellowish or brownish-yellow cross, gallbladder filling and the like can be seen during the dissection. The traditional Chinese medicine composition is accompanied with cockscomb pale and pale, muscular gastric ulcer, perforation of horny layer, small bleeding points in glandular stomach swelling, white dead spots in heart of a few chickens, congestion and bleeding phenomena in lung, yellow foam effusion in trachea, a few bleeding phenomena in intestinal tract, yellowish green mucus excrement, yellowish green liquid in muscular stomach, effusion or jelly-like effusion in abdominal cavity, yolk sac swelling or atrophy degeneration.
In view of the harm and the current situation of preventing and treating the chicken liver disease degeneration syndrome, how to select the medicine with high efficiency, convenient use, small toxic and side effect and no influence on human health to prevent and treat the disease becomes an important subject to be solved urgently.
According to the theory of Chinese veterinarian, liver is yang in yin, and is ascending and distributing in the main qi machine, the pathogenic factors of cold-dampness stagnates in the exterior, liver qi can not reach, yin essence can not go upwards and is retained in the interior, and yin essence is sticky with heat and is damp. Liver pertains to wood, which generates heart fire and liver qi stagnation, and can not supply heart yin, causing heart yin deficiency to generate internal heat. Liver diseases are first transmitted to the spleen, wood qi restricts earth, so that the yin essence of the spleen cannot ascend and is damp. Spleen qi is deficient and unable to nourish lung qi, so lung and trachea are dry and dry to become fistula. Spleen yang impairment, kidney yang impairment, lung-qi impairment, inability to nourish the seeds, and kidney qi restriction, resulting in atrophy, yellowing of the kidney and salting-out of uric acid. Therefore, it is seen that the five zang organs are affected one by one to form a vicious circle by taking liver injury as the initiation point and according to the relationship between generation and restriction.
Disclosure of Invention
The invention aims to provide the traditional Chinese medicine composition for preventing and treating the chicken liver disease syndrome, which has wide pharmacological action, small toxic and side effects, low pollution, low medicine residue and low medicine resistance and has good prevention and treatment effect on chicken liver injury.
The invention is realized by the following measures:
The traditional Chinese medicine composition for preventing and treating chicken hepatosis syndrome is characterized by comprising the following raw materials in parts by weight: main drugs: 0.1 to 10 portions of white peony root, 0.1 to 10 portions of bupleurum, 0.1 to 10 portions of immature bitter orange and 0.1 to 10 portions of liquorice.
The invention has the following specific characteristics:
The traditional Chinese medicine composition comprises the following raw materials in parts by weight: main drugs: 2.5 parts of white peony root, 2.5 parts of bupleurum root, 2.5 parts of immature bitter orange and 2.5 parts of liquorice.
the Chinese medicinal composition is prepared into any one of oral liquid, tablets, pills, capsules and granules.
The traditional Chinese medicine composition is prepared by the following method:
Taking the radix paeoniae alba, the radix bupleuri, the immature bitter orange and the liquorice according to the prescription amount, adding 1-12 times of water, decocting for 1-3 times, decocting for 0.5-2 h each time, filtering, combining the filtrates, concentrating the combined filtrates to the relative density of 1-2 (40-80 ℃), adding an appropriate amount of ethanol for alcohol precipitation to ensure that the alcohol content is 50% -80%, refrigerating for 12-36 h, filtering, recovering ethanol from the filtrate under reduced pressure, concentrating to an appropriate amount, adding an appropriate amount of potassium sorbate, filtering, filling and sterilizing to obtain the oral liquid.
The traditional Chinese medicine composition is prepared by the following method:
Taking the radix paeoniae alba, the radix bupleuri, the immature bitter orange and the liquorice according to the prescription amount, adding 1-12 times of water, decocting for 1-3 times, decocting for 0.5-2 h each time, filtering, combining the filtrates, concentrating the combined filtrates to the relative density of 1-2 (40-80 ℃), adding a proper amount of ethanol for alcohol precipitation to ensure that the alcohol content is 50-80%, refrigerating for 12-36 h, filtering, recovering the ethanol from the filtrates under reduced pressure and concentrating to a proper amount, then spray-drying the concentrated total extract in a spray-drying manner to obtain spray-dried powder, adding a proper amount of auxiliary materials into the spray-dried powder, and preparing into solid preparations such as tablets, pills, capsules, granules and the like.
The weight ratio of the potassium sorbate is 0.1-0.2%.
The traditional Chinese medicine composition is applied to preparation of medicines for treating chicken hepatosis syndrome.
The invention has the beneficial effects that: the traditional Chinese medicine composition has wide pharmacological action, small toxic and side effect, low pollution, low medicine residue and low drug resistance, and has good prevention and treatment effect on the chicken liver injury.
Drawings
Fig. 1 is a map of a white peony root standard substance in experiment two of the present specification.
FIG. 2 is a spectrum of a white peony root negative sample in experiment two of the present specification.
Detailed Description
In order to clearly illustrate the technical features of the present solution, the present solution is explained below by way of specific embodiments.
Example 1:
The traditional Chinese medicine composition for preventing and treating chicken hepatosis syndrome is characterized by comprising the following raw materials in parts by weight: main drugs: 2.5 parts of white peony root, 2.5 parts of bupleurum root, 2.5 parts of immature bitter orange and 2.5 parts of liquorice.
The invention has the following specific characteristics:
The traditional Chinese medicine composition is prepared by the following method:
Taking the radix paeoniae alba, the radix bupleuri, the immature bitter orange and the liquorice according to the prescription amount, adding 1-12 times of water, decocting for 1-3 times, decocting for 0.5-2 h each time, filtering, combining the filtrates, concentrating the combined filtrates to the relative density of 1-2 (40-80 ℃), adding an appropriate amount of ethanol for alcohol precipitation to ensure that the alcohol content is 50% -80%, refrigerating for 12-36 h, filtering, recovering ethanol from the filtrate under reduced pressure, concentrating to an appropriate amount, adding an appropriate amount of potassium sorbate, filtering, filling and sterilizing to obtain the oral liquid.
The traditional Chinese medicine composition is prepared according to the following method.
The traditional Chinese medicine composition is applied to preparation of medicines for treating chicken hepatosis syndrome.
Example 2
A traditional Chinese medicine composition for preventing and treating chicken hepatosis syndrome comprises the following raw materials in parts by weight: main drugs: 2.5 parts of white peony root, 2.5 parts of bupleurum root, 2.5 parts of immature bitter orange and 2.5 parts of liquorice.
The traditional Chinese medicine composition is prepared by the following method:
taking the radix paeoniae alba, the radix bupleuri, the immature bitter orange and the liquorice according to the prescription amount, adding 1-12 times of water, decocting for 1-3 times, decocting for 0.5-2 h each time, filtering, combining the filtrates, concentrating the combined filtrates to the relative density of 1-2 (40-80 ℃), adding a proper amount of ethanol for alcohol precipitation to ensure that the alcohol content is 50-80%, refrigerating for 12-36 h, filtering, recovering the ethanol from the filtrates under reduced pressure and concentrating to a proper amount, then spray-drying the concentrated total extract in a spray-drying manner to obtain spray-dried powder, adding a proper amount of auxiliary materials into the spray-dried powder, and preparing into solid preparations such as tablets, pills, capsules, granules and the like.
The weight ratio of the potassium sorbate is 0.2 percent.
Application of traditional Chinese medicine composition in preparation of medicine for treating chicken hepatosis syndrome
The traditional Chinese medicine composition for preventing and treating chicken hepatosis syndrome prepared by the test has no obvious change in appearance, identification, inspection and content inspection indexes during 24 months of storage, meets all regulations of quality standards of clinical prodrugs, and has basically stable quality.
The inventor of the medicine of the invention carries out a series of experimental researches in the aspects of selection, preparation method and application of raw material medicines, and determines the raw material medicines and the preparation method for preparing the medicine of the invention, so that the medicine of the invention has obvious outstanding curative effect advantages compared with the prior art.
Experiment I, research on preparation process of traditional Chinese medicine composition
1. Instruments and reagents
High performance liquid chromatography (HITACHI pump L-2130, detector L-2400; Agilent 1200 quaternary low pressure gradient pump, VWD detector); an air-blast drying box (digital display air-blast drying box GZX-9146 MBE); vacuum drying oven (model DZF-6050); spray drying SY-6000; granule WK-60; paeoniflorin reference (batch No. 110736-201438, purchased from China institute for food and drug testing, with the content of 96.4%); synephrine reference (lot No. 110727-201107, purchased from China institute for food and drug testing, in an amount of 99.4%); the reagents are chromatographically pure except methanol and acetonitrile, and the other reagents are analytically pure; wahaha purified water: hangzhou child Haha group Co., Ltd.
2. Method of producing a composite material
2.1 determination of extraction solvent
Firstly, according to the quality standards of white paeony root, Chinese thorowax root, immature bitter orange, liquorice and the like in the second part of the pharmacopoeia of the people's republic of China in 2010 edition, the purchased medicinal materials are respectively detected, and the medicinal materials meeting the requirements of the quality standards are subjected to the following experiments.
the method comprises the following steps of respectively extracting medicinal materials by using water, 50% ethanol and 80% ethanol as extraction solvents, wherein each extraction solvent is prepared in parallel by 3 parts, and the specific method comprises the following steps:
(1) The method takes water as an extraction solvent: weighing each medicinal material with the prescribed prescription amount, adding 25g of each medicinal material, adding 12 times of water, heating, decocting and extracting for 3 times, each time for 2h, filtering, mixing filtrates, concentrating to appropriate amount, adding appropriate amount of ethanol (making the ethanol content be 60%), precipitating with ethanol, refrigerating for 24 h, filtering, recovering ethanol from the filtrate under reduced pressure, and concentrating to 100ml to obtain the final product. The contents of paeoniflorin and synephrine were determined by HPLC, and the results are shown in Table 1.
(2) The method takes 50% ethanol as an extraction solvent: weighing each medicinal material with the prescribed prescription amount, adding 50% ethanol of 12 times of the amount of 25g of each medicinal material, heating, refluxing and extracting for 3 times, each time for 2h, filtering, mixing filtrates, concentrating under reduced pressure to recover ethanol and concentrating to appropriate amount, adding ethanol (making ethanol content be 60%), precipitating with ethanol, refrigerating for 24 h, filtering, recovering ethanol from filtrate under reduced pressure and concentrating to 100ml to obtain the final product. The contents of paeoniflorin and synephrine were determined by HPLC, and the results are shown in Table 1.
(3) The method takes 80% ethanol as an extraction solvent: weighing each medicinal material with the prescribed prescription amount, adding 80% ethanol of 12 times of the amount of 25g of each medicinal material, heating, refluxing and extracting for 3 times, each time for 2h, filtering, mixing filtrates, concentrating under reduced pressure to recover ethanol and concentrating to appropriate amount, adding ethanol (making ethanol content be 60%), precipitating with ethanol, refrigerating for 24 h, filtering, recovering ethanol from filtrate under reduced pressure and concentrating to 100ml to obtain the final product. The contents of paeoniflorin and synephrine were determined by HPLC, and the results are shown in Table 1.
TABLE 1 results of different extraction solvents
From the data in the table above in combination with the actual production, water was finally selected as the extraction solvent.
2.2 determination of Water extraction Process parameters
According to related documents and pre-test results, the decocting times, the decocting time and the water addition amount of medicinal materials are designed as main investigation factors, 3 levels are selected for each factor, see table 2, an L 9 (3 4) orthogonal table is selected for experiment, filtrate is collected and quantified, the content of paeoniflorin and synephrine is taken as an index, and the optimal extraction process is selected, and the results are shown in tables 3 and 4.
TABLE 2L 9 (3 4) orthogonal design factor horizon
Analysis of results
The result is shown in tables 3 and 4, 3 factors are visually analyzed and compared, R B R C R A can be seen, namely, the extraction of the traditional Chinese medicine composition is influenced most greatly, and the variance analysis result can be seen that the factor B (the number of times of decoction) is obviously different from the factor A (the time of decoction) and the factor C (the water addition amount) (P is less than 0.1). according to the experimental result, the factor A selects the best level 3, the factor B selects the best level 3, the factor C selects the best level 3, namely, A 3 B 3 C 3, but the factor A (the time of decoction) and the factor C (the water addition amount) are not obviously different, and the factor A, C selects the level 2 to be the best in consideration of factors such as large production and economy, so that the optimal extraction process of the traditional Chinese medicine composition is finally determined to be A 2 B 3 C 2, namely, the water is added by 10 times, 3 times, and each time is 1.5 hours, and the optimal extraction process is obtained.
TABLE 3 Water extraction orthogonal Experimental results
The total score ═ paeoniflorin content/maximum paeoniflorin content × 100 × 0.4+ (synephrine content/maximum synephrine content) × 100 × 0.4+ (dry extract yield/maximum dry extract yield) × 100 × 0.2.
TABLE 4 analysis of variance results
F(2,8)0.1＝3.110。
Verification test
The best process is verified: weighing 3 parts of medicinal materials according to the prescription amount, 100g of each medicinal material, adding 10 times of water into the medicinal materials according to the optimal extraction process, decocting for 3 times, 1.5h each time, performing tests, collecting filtrate and quantifying. The contents of paeoniflorin and synephrine were determined by HPLC, and the results are shown in Table 5.
Table 5 process verification results
As can be seen from Table 5, the paeoniflorin and synephrine have high content and uniform value, which indicates that the optimal process is stable and feasible.
2.3 investigation of Water extraction and alcohol precipitation Process
decocting with water to extract effective components of the medicine, and dissolving out ineffective components such as starch, mucus, protein, resin, tannin, etc., which are not removed, the oral liquid has poor clarity. Precipitates will be produced after the storage, which affects the quality. The ethanol precipitation method is adopted to remove the impurities, and the problem of loss of effective components after the ethanol precipitation is studied.
Preference of alcohol precipitation process
According to the results of the reference and the preliminary experiment, 3 factors of the concentration (A), the resting time (B) and the alcohol precipitation concentration (C) are selected as objects to be investigated, and the orthogonal experiment is carried out by adopting the L 9 (3 4) table by still using the contents of paeoniflorin and synephrine as evaluation indexes, wherein the level arrangement of the factors is shown in the table 6.
Table 6 alcohol precipitation process factor level table
alcohol precipitation process orthogonal test result
According to the table 6, 100g of the medicinal materials with the prescription amount and 9 parts of the medicinal materials are respectively weighed. According to the best extraction process, 10 times of water is added, the decoction is carried out for 3 times, the test is carried out for 1.5h each time, the extracting solution is filtered, the filtrate is mixed and concentrated to the concentrated solution with different initial densities specified in the table, absolute ethyl alcohol is added to the concentrated solution with specified alcohol precipitation concentration while stirring, the mixture is fully stirred and stands for specified time, the filtration is carried out, the filtrate is decompressed and the ethyl alcohol is recycled to 100ml, and the content of paeoniflorin and synephrine is respectively measured. The test results and the results of the anova are shown in tables 7 and 8.
TABLE 7 results of orthogonal experiments
Composite score ═ paeoniflorin content/maximum paeoniflorin content x 100 x 0.5+ (synephrine content/maximum synephrine content) x 100 x 0.5
TABLE 8 analysis of variance results
F(2,8)0.1＝3.110。
The result shows that the density of the factor A concentrated solution has significant difference on the test result, the primary and secondary sequence of all factors is R A R C R B, the level of the density 3 of the factor A concentrated solution is the best level 2 according to visual analysis, the best level 2 is selected for the factor B, and the best level 3 is selected for the factor C, namely A 2 B 2 C 3, but the levels of the factor B (rest time) and the factor C (alcohol precipitation concentration) have no significant difference, and the factor B B, C selects the level 2 as the best level by considering factors such as large production, economic indexes and the like, so that the optimal alcohol precipitation process of the traditional Chinese medicine composition is finally determined to be A 2 B 2 C 2, namely the alcohol precipitation concentration is 60%, the density of the concentrated solution is 1.08-1.12 (80 ℃), and the alcohol precipitation rest time is 24 hours.
Best process verification
The best process is verified: weighing 3 parts of medicinal materials according to the prescription amount, 100g of each medicinal material, adding 10 times of water into the medicinal materials according to the optimal extraction process, decocting for 3 times, 1.5h each time, performing tests, and collecting filtrate. Mixing the filtrates, concentrating to relative density of 1.08-1.12 (80 deg.C), adding appropriate amount of ethanol (to ethanol content of 60%), precipitating with ethanol, refrigerating for 24 hr, filtering, recovering ethanol from the filtrate under reduced pressure, and concentrating to 100 ml. The contents of paeoniflorin and synephrine were determined by HPLC, and the results are shown in Table 9.
Table 9 Process verification results
As can be seen from Table 9, the paeoniflorin and synephrine have high content and uniform value, which indicates that the optimal process is stable and feasible.
2.4 optimization of concentration Process
The extract obtained by extraction is concentrated after being filtered, and two different concentration methods of normal pressure concentration and reduced pressure concentration are compared. The results show that the reduced pressure concentration temperature is lower, so that more effective components can be retained, and the time is saved, therefore, the reduced pressure concentration is selected, and the table 10 shows.
TABLE 10 preferred results of the concentration Process
2.5 examination of drying methods
The drying process respectively inspects three drying modes of air-blast drying, reduced pressure drying and spray drying. Concentrating the extractive solution to relative density of 1.06(60 deg.C), taking out 20ml of sample, testing, dividing the rest sample into 4 parts, respectively air drying (2 parts), drying under reduced pressure and spray drying, recording drying time, determining dry extract water content by drying weight loss method, calculating effective constituent loss condition with the concentrated solution before drying as reference, see Table 11
Forced air drying (1): adopting a GZX-9146MBE digital display forced air drying oven, wherein the temperature is 80 ℃, and the time is about 6 hours; forced air drying (2): adopting a GZX-9146MBE digital display forced air drying oven, wherein the temperature is 95 ℃, and the time is 12 h; and (3) drying under reduced pressure: adopting a DZF-6050 type vacuum drying oven, adding phosphorus pentoxide at 50 ℃ to absorb water for about 4 hours; spray drying: a SY-6000 laboratory spray dryer is adopted, and the air inlet temperature is 170 ℃.
TABLE 11 drying Process investigation Table
As can be seen from the above table, the drying method is a key step that affects the content of the active ingredients, and the two drying methods of forced air drying and reduced pressure drying take longer time, and the loss of the active ingredients is higher, and the practice proves that: the higher the temperature, the longer the time, the greater the loss of the active ingredient. Spray drying to atomize the concentrated liquid, contacting with hot air, and vaporizing water to obtain dried product.
2.6 preparation of the formulations
Adding appropriate amount of adjuvants into the above spray-dried powder, and making into solid preparation such as tablet, pill, capsule, granule, etc. according to conventional preparation process.
Experiment two study on the method for measuring the content of traditional Chinese medicine in the invention
1. Instruments and reagents
Agilent 1260 high performance liquid chromatograph (G1311C quaternary pump, G1329B autosampler, G1316A column oven, G4212B diode array detector, Agilent chemical workstation); agilent 1200 hplc (equipped with G1322A degasser, G1311A quaternary pump, G1329A autosampler, G1315D diode array detector, Agilent chemical workstation). Paeoniflorin reference (batch No. 110736-201438, purchased from China institute for food and drug testing, with the content of 96.4%); acetonitrile and methanol are both chromatographically pure.
2 content determination method
2.1 preparation of the traditional Chinese medicine: the traditional Chinese medicine is prepared according to the following formula and preparation process.
2.1.1 prescription: 250g of white peony root, 250g of bupleurum root, 250g of immature bitter orange and 250g of liquorice
2.1.2 preparation process: adding 10 times of water into the four medicines, decocting for 3 times, and each time for 1.5h, collecting filtrates, mixing the filtrates, concentrating until the relative density is 1.08-1.12 (80 ℃), adding an appropriate amount of ethanol (the ethanol content is 60%), precipitating with ethanol, refrigerating for 24 h, filtering, recovering ethanol from the filtrate under reduced pressure, concentrating to 1000ml, adding potassium sorbate, filling, and sterilizing to obtain the oral liquid.
2.2 measurement of Paeoniflorin content
2.2.1 chromatographic conditions
Chromatographic conditions are as follows: a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile-0.05% phosphoric acid (14:86) as a mobile phase, and a C18 chromatographic column (250 mm. times.4.6 mm), with a column temperature of 30 ℃, a detection wavelength of 230nm, a flow rate of 1ml/min, and a sample volume of 10. mu.l. The theoretical plate number is not less than 3000 calculated according to paeoniflorin.
Preparation of control solutions: taking a proper amount of paeoniflorin reference substance, precisely weighing, and respectively adding 14% acetonitrile solution to obtain a solution containing 80 μ g of paeoniflorin per 1 ml.
preparation of a test solution: precisely measuring 1ml of the product, placing into a 50ml measuring flask, diluting with 14% acetonitrile to scale, shaking, filtering, and collecting the filtrate.
Preparation of white peony root negative sample solution: taking the medicinal materials of the other prescriptions except the white paeony root, and preparing the negative sample lacking the white paeony root according to the preparation process.
2.2.2 detection method
The control and negative sample solutions were measured precisely to 10. mu.L each, and injected into a high performance liquid chromatograph to perform detection, and the results are shown in FIG. 1.
Under the chromatographic condition, referring to the graph of a white paeony root negative sample in the attached figure 2, the position of the paeoniflorin peak (Rt is 12.756min) in the graph of the test sample is not interfered; the method for determining paeoniflorin has good specificity.
2.2.3 drawing of Standard Curve
Accurately weighing 20.24mg penoniflorin, placing into a 50ml measuring flask, adding 14% acetonitrile solution, dissolving, diluting, and metering to scale to obtain 401.76 μ g/ml control stock solution containing penoniflorin. Precisely measuring appropriate amount of control stock solution, and diluting with 14% acetonitrile to obtain serial control solutions containing paeoniflorin 16.07 μ g/ml, 40.18 μ g/ml, 80.35 μ g/ml, 160.70 μ g/ml, 200.88 μ g/ml, and 401.76 μ g/ml.
A series of control solutions containing paeoniflorin of 16.07 mu g/ml, 40.18 mu g/ml, 80.35 mu g/ml, 160.70 mu g/ml, 200.88 mu g/ml and 401.76 mu g/ml are analyzed according to established chromatographic conditions, a chromatogram and a peak area are recorded, linear regression is carried out by taking the peak area of the paeoniflorin as an ordinate and the concentration (mu g/ml) of the paeoniflorin as an abscissa, and a paeoniflorin standard curve (n is 6) is obtained, wherein y is 12.46x +21.14, r 2 is 0.999, and the linear relationship of the content of the paeoniflorin in the range of 16.07 mu g/ml to 401.76 mu g/ml is good.
2.2.4 precision
Precisely sucking 10 mu L of the same test solution, repeatedly injecting samples for 6 times, recording a chromatogram, calculating the average value of the peak area of paeoniflorin to be 1141.17, and calculating the relative standard deviation to be 0.87%, which indicates that the traditional Chinese medicine detection method has good precision.
2.2.5 repeatability
Preparing 6 parts of test solution in parallel according to the test solution preparation method; and respectively carrying out sample injection analysis according to the chromatographic conditions, recording a chromatogram, and calculating the content according to a standard curve. As a result: the RSD of the content of the paeoniflorin in the traditional Chinese medicine is 0.43 percent (n is 6). The repeatability of the traditional Chinese medicine detection method is good.
2.2.6 stability
Preparing a sample solution according to the sample solution preparation method for the same batch of samples, performing sample injection analysis at different times of 0, 2, 4, 6, 8, 10 and 12 hours respectively according to the chromatographic conditions, recording a chromatogram, and recording peak area values to investigate the in-day stability of the samples. As a result, the content of paeoniflorin was substantially stable within 12 hours, and the RSD was 0.37% as calculated by the paeoniflorin peak area. Indicating that the sample solution was stable within 12 hours.
2.2.7 sample recovery test
Precisely measuring 6 parts of sample with known content in 0.5ml, respectively placing in 50ml volumetric flasks, respectively adding 25ml paeoniflorin reference stock solution (90.89 mu g/ml), adding 14% acetonitrile, fixing volume to scale, preparing according to the preparation method of test solution, filtering the solution by a 0.45 mu m microporous filter membrane, sucking 10 mu l of sample injection, recording chromatogram, and calculating according to the peak area of an external standard method. The average recovery of paeoniflorin was 101.81%, and RSD was 0.73% (n-6). The sample recovery rate of the traditional Chinese medicine is good.
2.2.8 sample assay
The content of paeoniflorin in 10 batches of the traditional Chinese medicine of the invention is determined according to a proposed content determination method, and the result is shown in the following table 12.
TABLE 1210 measurement of the content of the sample batches
According to the technical guide principle of quality control research of veterinary traditional Chinese medicines and natural medicines, the content limit is determined according to 20 measurement data results of 10 batches of samples, the content condition of medicinal materials, the transfer rate under stable process conditions, and the stability test result of the preparation is considered, and the content limit is determined by combining the content condition of the medicinal materials and the actual condition of mass production.
The average content of paeoniflorin in 10 samples is 3.77mg/ml, and the average multiplied by 80% is 3.02 mg/ml. Therefore, we determined the content limit as the average of 10 batches of the test product content results multiplied by 80%.
The limit of content is 3.77mg/ml by 80% -3.02 mg/ml,
Finally, the content determination index in the draft is determined to be that the content of paeoniflorin (C 23 H 28 O 11) in the traditional Chinese medicine composition is not less than 3mg per 1 ml.
3. Conclusion
The method adopts high performance liquid chromatography to measure the content of paeoniflorin in the traditional Chinese medicine, other components have no interference on the measurement result, the methodological investigation result shows that the linear relation is good, and the method has good precision, recovery rate and repeatability and can be used for quality control of the product.
Experiment three experiments for preventing and treating liver injury by using traditional Chinese medicine composition
1 materials and methods
1.1 test drugs
The invention relates to an oral liquid.
the bifendate dripping pill is clinically used for patients with chronic persistent hepatitis and ALT elevation, and can also be used for ALT elevation caused by chemical poisons and medicaments.
1.2 test animals
SPF mice, weighing 18-20 g, half male and half female, purchased from southern university of medical science laboratory animal center, license number: SCXK (Yue) 2011-. The complete feed is fed to mice during the test period, the room temperature is controlled to be 19-25 ℃, and the relative humidity is 30% -70%.
1.3 Main instruments
BS-380 automatic biochemical analyzer; model 1416R high speed refrigerated centrifuge; t3000 electronic balance.
1.4 test methods
72 SPF mice are randomly divided into 6 groups, namely a blank control group, a model group, a positive control group (150mg/kg) and low, medium and high dose groups (100, 200 and 400mg/kg) of the oral liquid, and each group contains 12 mice. The positive control group is administered with bifendate dripping pill 1 time per day for 7 days; the blank control group and the model group were perfused with the same volume of physiological saline.
After 2h of the last administration, except a blank control group (injected with olive oil), 10ml/kg of 0.1 percent CCl 4 olive oil solution is injected into the abdominal cavity of other groups, and a mouse acute liver injury model is established.
1.5 measurement index
1.5.1 serum Biochemical indicators
After a mouse acute liver injury model is established for 16 hours, 10 mice (each half of male and female) are randomly extracted from each group, eyeballs are picked for blood sampling, serum is separated, and the levels of alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) are measured by a full-automatic blood biochemical analyzer (or an ELISA kit).
1.5.2 liver tissue oxidase index
After a mouse acute liver injury model is established for 16 hours, 10 mice (each half of a male and a female) are randomly selected from each group, subjected to autopsy, cut off part of liver tissues, wiped and dried by filter paper, weighed, added with normal saline to prepare 10% liver tissue homogenate, centrifuged, and then supernatant is taken, and the SOD and MDA activities are measured according to the kit specification.
1.5.3 organ indices
After a mouse acute liver injury model is established for 16 hours, 12 mice (each half of male and female) are taken from each group, the weight is weighed, killed, hearts, livers, spleens, lungs and kidneys are taken, the weights are weighed, and the organ indexes are calculated. Organ index is organ weight (mg)/body weight (g).
1.5.4 dissection and histological observation
After a mouse acute liver injury model is established for 16 hours, 10 mice (each half of a female and a male) are randomly extracted from each group, dissected, cut part of liver tissues, fixed by 10% neutral formalin, sliced by paraffin, stained by HE, and observed by a microscope for pathological changes.
1.6 data processing
Data are presented as mean ± standard deviation, and single-factor analysis of variance was performed using SPSS 20.0 software analysis to compare the significance of differences in each group.
2. results
2.1 serum ALT, AST Activity changes
The activity changes of serum ALT and AST of each group of mice are shown in a table 13, glutamic-oxaloacetic transaminase (ALT) and glutamic-pyruvic transaminase (AST) of each CCl 4 injury group are obviously higher than those of a blank group, compared with a model group, the oral liquid can obviously reduce the ALT and AST content of the serum (P <0.05) when being administrated at different concentrations, and the oral liquid has no obvious difference (P >0.05) between the oral liquid and a positive control group when being administrated at high dose.
TABLE 13 serum ALT and AST Activity changes in groups of mice
Note that a-d data in the same column indicate significant differences between letters (P < 0.05). The following table is given below.
2.2 Activity Change of SOD and MDA in liver homogenate
the activity changes of SOD and MDA in liver homogenate of each group of mice are shown in a table 14, the SOD level in the liver homogenate of each CCl 4 damage group is obviously lower than that of a blank group, and compared with a model group, the oral liquid with three concentrations after being drenched can improve the SOD level in the liver homogenate, wherein the dosage group in the oral liquid is obviously higher than that of the model group (P is less than 0.05), the MDA level in the liver homogenate of each CCl 4 damage group is obviously higher than that of the blank group, and compared with the model group, the oral liquid with three concentrations after being drenched can obviously reduce the MDA level in the liver homogenate (P is less than 0.05).
TABLE 14 variation of SOD and MDA activities in liver homogenates of mice in each group
2.3 organ index changes in mice of each group
The change of the organ index of each group of mice is shown in Table 15, the heart index, the spleen index, the lung index and the kidney index of each group of mice have no obvious difference (P >0.05), and the liver index of each model group is increased after the CCl 4 of the mice is established for acute liver injury, wherein the model group is the largest and is obviously higher than a blank control group (P <0.05), while the liver index of the oral liquid of the invention in low, medium and high doses and the liver index of the mice in the positive control group have no obvious difference (P >0.05) with the blank control group.
TABLE 15 Change in organ index of mice in each group
2.4 hepatic histology changes
The normal control group takes the central vein as the axis and is arranged in a radial shape, the liver cord is arranged normally, the liver cells are polygonal, the nucleus is large and round, and the structure is normal; the liver slices of the mice in the model group show disorder of liver cord arrangement, sinus clearance and central venous dilatation congestion and partial hepatocyte degeneration; the liver tissues of mice in the oral liquid low, medium and high dose administration groups are close to those of a normal control group, and other obvious pathological changes are not seen except occasional liver cells which are slightly swollen. 3. Conclusion
After a CCl 4 induced mouse acute liver injury model is established, serum ALT and AST of each injury group are obviously increased, SOD in liver homogenate is obviously reduced, MDA activity is increased, liver index is obviously increased, the establishment of the mouse acute liver injury model is successful, the results of each administration group are compared, the liver index of the mouse with low, medium and high doses and a positive control group is not obviously different from that of a blank control group (P is more than 0.05), compared with the model group, the serum ALT and AST contents of the mouse with medium and high doses can be obviously reduced by infusing the oral liquid disclosed by the invention (P is less than 0.05), the serum ALT and AST contents of the mouse with medium and high doses are not obviously different from that of the positive control group (P is more than 0.05), compared with the model group, the oral liquid disclosed by the invention with different concentrations can improve the SOD level in liver, wherein the dosage group disclosed by the oral liquid disclosed by the invention is obviously higher than that of the model group (P is less than 0.05), compared with the model group, the oral liquid disclosed by the invention, the oral liquid disclosed by the invention, the oral liquid disclosed by the invention, the oral liquid disclosed by the invention.
Experiment III the oral liquid of the invention is used for treating mice acute liver injury caused by AFB1
1. Materials and methods
1.1 test drugs
The oral liquid of the invention;
Bifendate dripping pill;
Aflatoxin B1(AFB 1).
1.2 test animals
SPF mice, 18-20 g weight, half male and half female. The complete feed is fed to mice during the test period, the room temperature is controlled to be 19-25 ℃, and the relative humidity is 30% -70%.
1.3 Main instruments
Model 1416R high speed refrigerated centrifuge (zhhai black horse medical instruments ltd); model T3000 electronic balance (double jie test instruments factory, department of orthodox city).
1.4 Primary reagents
Alanine Aminotransferase (ALT) kit;
Aspartate Aminotransferase (AST) kits;
A SOD kit;
malondialdehyde (MDA) assay kit;
total protein quantification test kit.
1.5 test methods
1.5.1 basis of dosage of molding medicine
AFB1 is a highly hepatotoxic and carcinogenic mycotoxin, is ubiquitous in soil, various foods and animal feeds, and seriously threatens human and animal health, short-term exposure can cause acute liver injury such as hepatocyte necrosis, toxic hepatitis, bile duct hyperplasia, immunosuppression and the like of human and animals, and long-term exposure can even cause liver cancer, human [4] such as Torreyan and the like induces acute AFB1 liver injury of rats by single-dose intragastric administration of 2mg/kg bw AFB1 (dissolved in DMSO), a blank control group is given with an equal amount of DMSO, and a rat acute liver injury model is successfully established.
1.5.2 test animal grouping and handling
120 SPF mice are taken and randomly divided into 6 groups, namely a blank control group, a model group, a positive control group (bifendate, 150mg/kg) and low, medium and high dose groups (1, 2 and 4mL/kg) of the oral liquid, wherein each group contains 20 mice (half of male and female). Except for the blank group, each group was induced to acute AFB1 liver injury by single-dose intragastric gavage with 2 mg/kg-bw of AFB1 (dissolved in DMSO) 0.4mL per day, and the blank group was given an equal amount of DMSO once a day for 3 consecutive days to establish a liver injury model. After modeling, 045.4mL of corresponding drugs are infused into the stomach of each group according to the corresponding weight, and the same amount of physiological saline is infused into the stomach of mice of the blank group and the model group once a day for 7 days continuously. Samples were taken from 10 mice (male and female halves) per group at 3d and 7d post-dose, respectively.
1.6 measurement of indices
1.6.1 serum Biochemical indicators
After administration for 3d and 7d, 10 mice (male and female halves) were randomly selected per group, and blood was collected from the eyeballs, and serum was separated, and ALT and AST levels were measured using ALT and AST kits.
1.6.2 liver tissue oxidase index
After administration for 3d and 7d, 10 mice (male and female halves) were randomly selected from each group, dissected, partially harvested, wiped with filter paper, weighed, homogenized with physiological saline solution to 10% liver tissue, centrifuged, and the supernatant was collected, and SOD and MDA activities were measured according to the kit instructions.
1.6.3 organ indices
After 7 days of administration, 10 mice (male and female halves) were taken from each group, weighed, and subjected to a autopsy to obtain the heart, liver, spleen, lung, and kidney, respectively, and the organ index was calculated. Organ index is organ weight (mg)/body weight (g).
1.6.4 dissection and histological observation
After 7 days of administration, 10 mice (female and male halves) were randomly selected from each group, dissected, a part of liver and kidney tissues was excised, fixed in 10% neutral formalin, paraffin sectioned, HE stained, and pathological changes were observed under a microscope. 2. Results
2.1 serum ALT, AST Activity changes
The serum ALT and AST activity changes of each group of mice are shown in Table 16 and Table 17. From table 16, the ALT activity of each AFB1 model group is significantly higher than that of the blank group (P <0.05), the AST activity is not significantly different from that of the blank group (P >0.05), and compared with the model group, the ALT and AST contents in serum can be significantly reduced after the oral liquid of the invention with different concentrations is infused for 3d (P < 0.05); the serum AST and ALT contents of mice administrated with the oral liquid of the invention with three concentrations have no significant difference with a positive control group (P is more than 0.05).
From table 17, after the oral liquid of the invention is administrated for 7d, the ALT activity of each AFB1 model group is obviously higher than that of a blank group (P <0.05), AST has no obvious difference (P >0.05) with the blank group, and compared with a model group, the ALT and AST contents of blood serum (P <0.05) can be obviously reduced after the oral liquid of the invention with three doses is administrated for 7 d; after the oral liquid of the invention is administrated for 7 days, the ALT activity of the serum has significant difference (P <0.05) with the middle dose group, the high dose group and the positive control group, and the AST content in the serum of each treatment group has no significant difference (P > 0.05);
Table 16 administration of 3d groups of mice with changes in serum ALT and AST activities (n ═ 10)
Note: the data in the same column are marked with different letters to indicate significant difference, i.e., P < 0.05. The following table is the same.
Table 17 administration of ALT and AST activity changes in 7d groups of mice (n is 10)
2.2 Activity Change of SOD and MDA in liver homogenate
The changes in SOD and MDA activities in liver homogenates of the mice of each group are shown in tables 18 and 19. From table 18, the SOD levels in liver homogenates of each AFB1 model group were significantly lower than those of the blank group, and compared with the model group, the SOD levels in liver homogenates (P <0.05) were significantly improved by the oral liquid of the present invention administered at three concentrations; the MDA level in liver homogenate of each AFB1 model mouse is not significantly different from that in a blank group (P is greater than 0.05), and compared with the model group, the oral liquid with three concentrations can significantly reduce the MDA level in the liver homogenate of the mouse (P is less than 0.05).
From table 19, after the oral liquid of the invention is perfused for 7 days, the SOD level in liver homogenate of each treatment group has no significant difference (P >0.05) with that of a blank group and has significant difference (P <0.05) with that of a model group; compared with the model group, each treatment group can obviously reduce the MDA level (P <0.05) in the liver homogenate of the mice, and the MDA level in the liver homogenate of the mice of the low-dose group and the high-dose group of the oral liquid has no obvious difference (P >0.05) from that of the blank group.
Table 18 administration of SOD and MDA activity changes in liver homogenates of 3d groups of mice (n ═ 10)
table 19 administration of SOD and MDA activity changes in liver homogenates of 7d groups of mice (n ═ 10)
2.3 organ index changes in mice of each group
The change in organ index of each group of mice is shown in table 20. From table 20, no significant difference was observed in the heart, lung and kidney indices of the 7 d-dosed groups, while the liver index and spleen index of the model group were significantly different (P <0.05) compared to the blank group, and no significant difference was observed between the treated group and the blank group (P > 0.05).
Table 20 administration of 7d changes in organ index (n ═ 10) in each group of mice
3. Conclusion
After the mice are drenched with AFB1, compared with a blank group, the serum AST and ALT contents of the mice in the model group are obviously improved, and the activities of the liver homogenate SOD and the MDA of the mice are also obviously different from those in the blank group, which indicates that the establishment of the model of the mice acute liver injury caused by AFB1 is successful.
After the medicine is taken for 7d, the results of all administration groups are compared, compared with a model group, the oral liquid with three concentrations can obviously reduce the ALT and AST contents of the mouse serum (P <0.05), the ALT contents of the mouse serum with middle and high doses in the administration do not have obvious difference (P >0.05) with the positive control group mouse serum, and the AST contents of the mouse serum with three concentrations in the administration do not have obvious difference (P >0.05) with the positive control group mouse serum; compared with a model group, the oral liquid of the invention with three concentrations can obviously improve the SOD level (P is less than 0.05) in the liver homogenate of the mouse; compared with a model group, the oral liquid of the invention with three concentrations can obviously reduce the MDA level (P is less than 0.05) in liver homogenate of mice. No significant difference exists in the heart index, the lung index and the kidney index of each group after 7d administration, the liver index and the spleen index of the model group are significantly different compared with the blank group (P <0.05), and the liver index and the spleen index of each treatment group are not significantly different from the blank group (P > 0.05). The result shows that the oral liquid has better treatment effect on acute liver injury caused by mouse AFB 1.
Experiment four clinical trials of the oral liquid for treating chicken liver injury
1 materials and methods
1.1 test materials
1.1.1 test drugs and reagents
The oral liquid of the invention;
Control drug, Long Dan Xiegan san.
1.1.3 test apparatus
BS-380 automatic biochemical analyzer.
1.2 test methods
1.2.1 introduction of disease cases in laboratory chickens in clinical farming
1.2.1.1 onset and clinical symptoms
a certain breeding farm technician reflects that about 2600 slow-growing type Sanhuang chickens of 50 days old in the chicken farm begin to get ill before 10 days, part of the chickens show lethargy and anorexia, and later the chickens show asthenia, anemia, pale coronaria, weight loss, diarrhea and bloody stool, the sick chickens mostly die due to exhaustion of the dead appetite, the disease course is long, more than 20 chickens die each day on average, the number of the dead chickens is increased day by day after the disease starts to get ill before 10 days, and the disease has no effect after 7 days of treatment by using various antibiotics.
The method comprises the steps of inquiring a breeder party in charge of the chicken house in a farm to know that a part of feed in the farm is drenched by heavy rain, and finding out that the drenched feed is mainly supplied to the sick chicken crowd through investigation.
1.2.1.2 Caesarean Change
The cesarean-examination of dead chickens mainly shows emaciation, bleeding spots in subcutaneous muscles, hepatomegaly and necrotic spots, and most chickens show symptoms such as pericarditis, peritonitis, perihepatitis, air sacculitis and the like.
1.2.1.3 differential diagnosis
According to investigation, the farm can immunize chicken newcastle disease and bird flu, in order to further confirm, the sick chicken of the sick chicken group are selected and inspected, a hemagglutination inhibition experiment is carried out to monitor antibodies against the chicken newcastle disease and the bird flu, and test results show that the antibody titer of the sick chicken group is between 6 log2 and 8 log2, so that the infection possibility of the chicken newcastle disease and the bird flu virus can be eliminated.
1.2.1.4 laboratory diagnostics
The aflatoxin assay is carried out by using an aflatoxin B 1 kit rapid diagnosis method, the detection result shows that the feed contains 250 mug/kg of aflatoxin, and the content of aflatoxin is seriously exceeded, meanwhile, serum of sick chickens is sampled and detected as glutamic oxaloacetic transaminase (AST) and glutamic pyruvic transaminase (ALT), and the detection result shows that the serum of glutamic oxaloacetic transaminase (AST) and glutamic pyruvic transaminase (ALT) of the sick chickens are obviously higher than indexes of other healthy chickens of the same age of the farm.
According to the diagnosis result in the laboratory, the sick chicken flocks are preliminarily judged to be aflatoxin poisoning.
1.2.1.5 comprehensive diagnostic results
By investigating the feeding process of the sick chicken flocks, the clinical symptoms and anatomical symptoms of the sick chicken, the developed differential diagnosis and laboratory detection diagnosis, and simultaneously combining the clinical medication treatment results during the disease period, the sick chicken flocks can be confirmed to be aflatoxin poisoning after comprehensive analysis.
1.2.2 Experimental animals grouping and handling
Approximately 2400 live chickens described in 1.2.1 were equally divided into 3 columns, each column being taken as 1 test group, i.e. about 800 experimental chickens per group, while all experimental chickens were replaced with feed (i.e. fed the same feed as the other healthy chickens in the farm). Group 1 is blank control group (no treatment), group 2 is controlled by using control veterinary drug (gentian liver-fire-purging powder), group 3 is controlled by using oral liquid, the drug is administered for 7 days, and the observation is carried out for 7 days after the drug is stopped, namely the test period is 14 days, and the specific grouping and treatment conditions are shown in table 21. The experimental chickens were fed and drunk freely throughout the entire experimental period, and otherwise were following routine procedures.
TABLE 21 Experimental grouping and treatment
1.3 detection indexes in the test
1.3.1 clinical signs
During the experiment, the clinical manifestations of the experimental chicken such as ingestion, drinking and diarrhea are carefully observed every morning and afternoon, and the number of dead experimental chickens is recorded.
1.3.2 Biochemical index of blood
On the 0 th day, the 4 th day and the 7 th day of the test, 12 test chickens were randomly selected from each test group, blood serum was separated after blood collection, and blood biochemical indexes such as serum glutamic-oxaloacetic transaminase (AST) and glutamic-pyruvic transaminase (ALT) were detected.
1.3.3 growth Performance of Experimental Chicken
Before, during and after the experiment, 40 experimental chickens are randomly selected from 3 experimental groups respectively and weighed, the average weight and the weight gain of each group of experimental chickens are calculated, and the feed conversion ratio of the experimental chickens is calculated.
1.4 data analysis
The test data are expressed as (mean ± sd), and statistical analysis and processing are performed using SPSS statistical analysis software.
2. results and analysis
2.1 clinical presentation
After the sick chicken flock is diagnosed to be aflatoxin poisoning according to the test, after the feed is replaced for the sick chicken flock (namely the feed is fed to the sick chicken flock with the same feed as other healthy chickens in a farm), the clinical symptoms of the 1 st to 3 th groups of sick chicken flock are improved day by day, and the number of dead sick chickens is reduced day by day during the test. Meanwhile, the test results also show that the clinical performance of the experimental chickens in the groups 2 and 3 which are respectively dosed with the medicine for prevention and treatment is better than that of the experimental chicken in the group 1, and the comparison test results (see table 22) show that the total death number of the experimental chickens in the groups 2 and 3 is obviously lower than that of the experimental chicken in the group 1 (35) and the group 3 is lower than that of the experimental chicken in the group 2 during the dosing period of the experiment (the experimental days 1-7); during the observation period after the test dosing is finished (test days 8-14), the total death number of experimental chickens in the groups 2 and 3 is obviously lower than that in the group 1 (15), and the group 3 is lower than that in the group 2.
TABLE 22 experimental period of chicken death in each group
Note: the test results are divided into two test stages of tests 1 to 7d and tests 8 to 14d for statistics.
The test results show that when the oral liquid is used for preventing and treating the aflatoxin poisoning disease which is mainly diseased due to liver injury, the clinical symptoms of sick chicken can be obviously improved, the death number of the sick chicken is reduced, and the sick chicken can be promoted to be better recovered.
2.2 Biochemical index of blood
The test results showed (see table 23) that the differences in serum AST levels were not significant in the experimental chickens before the administration of the test, and after 4 and 7 days of administration, the AST levels in the experimental chickens in group 3 treated with oral liquid were significantly lower than in group 1 of the blank control (P <0.01), and group 3 was also lower than in group 2 treated with the control veterinary drug but did not significantly differ (P > 0.05).
TABLE 23 Experimental Chicken AST Change
Note: the data in the same column with different shoulder marks in lower case letters shows significant difference (P <0.05), and the data in the same column with different shoulder marks in upper case letters shows significant difference (P < 0.01); the letters are identical or have no letter shoulder marks with no significant difference (P >0.05), and the following is the same.
The test results showed (see table 24) that the serum ALT levels of the experimental chickens were not significantly different from each other before the administration of the test, and after 4 and 7 days of administration, the ALT levels of experimental chickens in group 3 treated with oral liquid were significantly lower than those in group 1 of the blank control (P <0.01), and group 3 was also lower than those in group 2 of the control veterinary drug but did not significantly differ (P > 0.05).
TABLE 24 ALT Change in Experimental Chicken
The test results show that the oral liquid is added into the aflatoxin poisoning experimental chicken drinking water, so that the clinical symptoms of sick chickens can be relieved to a certain extent by reducing the AST and ALT levels of the experimental chickens, the liver function of the whole sick chicken group is effectively protected, and the sick chicken group can better tolerate the oral liquid and gradually recover health; the test result also shows that the prevention and treatment effect of the oral liquid on the aflatoxin poisoning chicken flocks is slightly better than that of a control veterinary drug, but the difference is not obvious.
2.3 growth Performance of the experimental chicks
The test results show (see table 25) that the weight difference of the experimental chickens in each group is not significant before the test administration, and after 4 and 7 days of the administration, the weight average of the experimental chickens in the 3 rd group which are controlled by using the oral liquid is significantly higher than that of the experimental chickens in the 1 st group of the blank control group (P < 0.05; P <0.01), and the weight average of the experimental chickens in the 3 rd group which are also higher than that of the experimental chickens in the 2 nd group which are controlled by the control veterinary drug is not significant (P > 0.05).
TABLE 25 Experimental changes in chicken body weight
the test results show (see table 26), that the average weight gain of experimental chickens in group 3 which are controlled by using the oral liquid is obviously higher than that of the blank control group 1 during the whole test period, but the feed-weight ratio is obviously lower than that of the group 3; meanwhile, the test result also shows that the average weight gain of experimental chickens in the group 3 is higher than that of the group 2 controlled by the control veterinary drug, but the feed weight ratio of the experimental chickens in the group 3 is lower than that of the group 2.
TABLE 26 Experimental Chicken feed weight ratio during the test period
The test results show that the oral liquid is added into drinking water of experimental chickens, so that the adverse effect of aflatoxin on the growth performance of diseased chickens can be effectively relieved, the growth of the experimental chickens is effectively promoted, and the feed-weight ratio is reduced; the test result also shows that the prevention and treatment effect of the oral liquid on the aflatoxin poisoning experimental chicken flocks is slightly better than that of a control veterinary drug, but the difference is not obvious.
3. Conclusion
In this test, after the sick chicken flocks were fed with the feed (i.e., the same feed as the other healthy chickens in the farm) after the sick chicken flocks were diagnosed as the sick chicken flocks with aflatoxin poisoning, the clinical symptoms of all 3 sick chicken flocks were improved day by day, and the number of the dead sick chickens during the test period was reduced day by day. And the comparative test results show that the number of dead chickens of the experimental chickens of the groups 2 and 3 which are controlled by the drug administration in the test period is obviously lower than that of the blank control group 1, the serum AST and ALT levels of the experimental chickens of the groups 2 and 3 are obviously lower than that of the group 1 (P <0.01), the body weights of the experimental chickens are obviously higher than that of the group 1 (P < 0.05; P <0.01), the material weight ratio of the groups 2 and 3 is obviously lower than that of the group 1, but the difference of the results between the groups 2 and 3 is not obvious.
The comprehensive analysis of the test results shows that the oral liquid with the concentration of 0.5 percent is added into drinking water of experimental chickens, so that the effect of the oral liquid on preventing and treating aflatoxin poisoning of the experimental chickens is better, and the effect of the oral liquid is slightly better than that of a control veterinary drug, but the difference is not obvious.
The technical features of the present invention which are not described in the above embodiments may be implemented by or using the prior art, and are not described herein again, of course, the above description is not intended to limit the present invention, and the present invention is not limited to the above examples, and variations, modifications, additions or substitutions which may be made by those skilled in the art within the spirit and scope of the present invention should also fall within the protection scope of the present invention.

Claims (7)

1. The traditional Chinese medicine composition for preventing and treating chicken hepatosis syndrome is characterized by comprising the following raw materials in parts by weight: main drugs: 0.1 to 10 portions of white peony root, 0.1 to 10 portions of bupleurum, 0.1 to 10 portions of immature bitter orange and 0.1 to 10 portions of liquorice.
2. The traditional Chinese medicine composition for preventing and treating chicken liver disease syndrome according to claim 1, which is characterized by comprising the following raw materials in parts by weight: main drugs: 2.5 parts of white peony root, 2.5 parts of bupleurum root, 2.5 parts of immature bitter orange and 2.5 parts of liquorice.
3. The Chinese medicinal composition for preventing and treating the chicken liver disease syndrome according to claim 2, wherein the Chinese medicinal composition is prepared into any one of oral liquid, tablets, pills, capsules and granules.
4. The traditional Chinese medicine oral liquid for treating chicken liver disease syndrome according to claim 3, wherein the traditional Chinese medicine composition is prepared by the following method:
Taking the radix paeoniae alba, the radix bupleuri, the immature bitter orange and the liquorice according to the prescription amount, adding 1-12 times of water, decocting for 1-3 times, decocting for 0.5-2 h each time, filtering, combining the filtrates, concentrating the combined filtrates to the relative density of 1-2 (40-80 ℃), adding an appropriate amount of ethanol for alcohol precipitation to ensure that the alcohol content is 50% -80%, refrigerating for 12-36 h, filtering, recovering ethanol from the filtrate under reduced pressure, concentrating to an appropriate amount, adding an appropriate amount of potassium sorbate, filtering, filling and sterilizing to obtain the oral liquid.
5. The traditional Chinese medicine oral liquid for treating chicken liver disease syndrome according to claim 3, wherein the traditional Chinese medicine composition is prepared by the following method:
Taking the radix paeoniae alba, the radix bupleuri, the immature bitter orange and the liquorice according to the prescription amount, adding 1-12 times of water, decocting for 1-3 times, decocting for 0.5-2 h each time, filtering, combining the filtrates, concentrating the combined filtrates to the relative density of 1-2 (40-80 ℃), adding a proper amount of ethanol for alcohol precipitation to ensure that the alcohol content is 50-80%, refrigerating for 12-36 h, filtering, recovering the ethanol from the filtrates under reduced pressure, concentrating to a proper amount, spray-drying the concentrated total extract in a spray-drying manner to obtain spray-dried powder, adding a proper amount of auxiliary materials into the spray-dried powder, and preparing into solid preparations such as tablets, pills, capsules, granules and the like.
6. The traditional Chinese medicine composition for preventing and treating chicken liver disease syndrome according to claim 5, wherein the weight ratio of potassium sorbate is 0.1-0.2%.
7. The traditional Chinese medicine composition for preventing and treating chicken hepatosis syndrome according to claim 1, wherein the traditional Chinese medicine composition is used for preparing a medicine for treating chicken hepatosis syndrome.
CN201910697244.2A 2019-07-30 2019-07-30 Traditional Chinese medicine composition for preventing and treating chicken hepatosis syndrome as well as preparation method and application thereof Pending CN110548079A (en)

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