CN115634245B - Veterinary medicine composition and decoction for preventing and treating liver injury of livestock and poultry and application of veterinary medicine composition and decoction - Google Patents
Veterinary medicine composition and decoction for preventing and treating liver injury of livestock and poultry and application of veterinary medicine composition and decoction Download PDFInfo
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Abstract
The invention discloses a veterinary medicine composition, a decoction thereof, a preparation method and application thereof, wherein the composition is prepared from the following raw materials in parts by weight, 20-40 parts of loosestrife, 15-20 parts of Hangzhou chrysanthemum and 25-35 parts of thalictrum hemipterum. The target organ for which the decoction formed by the veterinary drug composition aims is liver, has repairing and treating effects on various forms of acute liver injury, and is applied to providing reference data and data support for the medication of liver injury diseases in livestock and poultry farming.
Description
Technical Field
The invention relates to the field of veterinary medicines, in particular to a veterinary medicine composition for preventing and treating liver injury of livestock and poultry, a decoction and application thereof.
Background
The herba Phyllanthi is root or whole plant of Phyllanthus niruri of Phyllanthus of Prinsepiaceae, and has bitter and pungent taste and mild nature, and enters liver and spleen meridians. Has effects of clearing heat, promoting diuresis, promoting blood circulation, removing blood stasis, removing toxic substances, and resolving carbuncle, and can be used for treating edema, heat stranguria, jaundice, dysentery, rheumatic arthralgia, leukorrhagia, amenorrhea, traumatic injury, fracture, traumatic hemorrhage, acute mastitis, furuncle, and snake bite.
The chrysanthemum is a perennial herb, and according to medical analysis, the chrysanthemum contains chrysanthemum city, glance sideways at parts of amino acid, trace vitamins and other components, and has slightly cold nature and sweet and bitter taste. Has effects of dispelling pathogenic wind, clearing heat, removing liver fire, improving eyesight, removing toxic substances, and relieving inflammation, and can be used for treating hypertension, migraine, acute conjunctivitis, etc.
Thalictrum is root and rhizome of Thalictrum planatum of Thalictrum of Ranunculaceae, often used as a Tibetan medicine, enters stomach, large intestine and liver channels, has the effects of clearing heat and drying dampness, purging fire and removing toxin, and is mainly used for treating damp-heat diarrhea, jaundice, leucorrhea, toothache due to wind-fire, conjunctival congestion and swelling pain, and pyocutaneous disease and toxic swelling.
The composition formed by the three medicaments and the therapeutic effect thereof are not disclosed in the prior art.
Disclosure of Invention
The invention aims to provide a veterinary medicine composition and decoction for preventing and treating liver injury of livestock and poultry and application thereof.
The invention provides the following technical scheme:
a veterinary medicine composition for preventing and treating liver injury of livestock and poultry comprises the following components in parts by mass: 20-40 parts of loosestrife, 15-20 parts of chrysanthemum morifolium ramat and 25-35 parts of thalictrum hemipterum.
Also provides a decoction prepared from the veterinary medicine composition.
Further, the decoction prepared from the veterinary medicine composition contains the following active ingredients: rutin, luteolin, isochlorogenic acid A and berberine hydrochloride.
The invention also provides a preparation method of the decoction prepared from the veterinary drug composition, which comprises the following steps:
s1, adding sterilized deionized water into the components for soaking, and then decocting for one or more times to obtain decoction;
s2, concentrating the decoction until the concentration of the solid matters is 1-5 g/ml, thereby obtaining the decoction.
Further, the solid-to-liquid ratio of the components to the sterilized deionized water is 1:10-1:20.
Further, the temperature of the decoction is 55-75 ℃, and the time of each decoction is 30-50 min;
the invention also provides application of the veterinary drug composition or the decoction prepared by the veterinary drug composition in preparation of veterinary drugs for preventing and treating acute liver injury.
The invention also provides application of the veterinary drug composition or the decoction prepared by the veterinary drug composition in preparation of veterinary drugs for preventing and treating chemical acute liver injury caused by carbon tetrachloride.
The invention also provides application of the veterinary drug composition or the decoction prepared by the veterinary drug composition in preparing medicines for preventing and treating acute drug-induced liver injury caused by acetaminophen.
The invention also provides application of the veterinary drug composition or the decoction prepared by the veterinary drug composition in preparation of veterinary drugs for preventing and treating alcoholic acute liver injury caused by alcohol.
The invention has the following beneficial effects:
the decoction formed by the composition has the effects of repairing and treating various forms of acute liver injury, has a better protective effect on liver injury caused by excessive use of calf yak praziquantel in a specific clinical test, can effectively reduce serum levels of glutamic pyruvic transaminase and glutamic oxaloacetic transaminase, and can be further used as a medicament for preventing and treating liver injury diseases caused by excessive use of medicaments in livestock and poultry farming industry.
The composition has good oxidation resistance, bacteriostasis and anti-inflammatory property, and is nontoxic and harmless to livestock and poultry and environment.
Drawings
Fig. 1 is an HPLC diagram of the mixed standard provided in example 1.
Fig. 2 is an HPLC diagram of the composition decoction provided in example 1.
FIG. 3 is a toxicity test organ section (HE staining, ×400) provided in example 1.
FIG. 4 is a CCl provided in example 1 4 And (5) an appearance change chart of the acute liver injury.
FIG. 5 is a CCl provided in example 1 4 The resulting acute liver injury was a liver histopathological change (HE staining, ×400).
FIG. 6 is a CCl provided in example 1 4 The serum biochemical index detection result of the mice with acute liver injury is obtained.
FIG. 7 is a CCl provided in example 1 4 The liver antioxidant capacity of the mice with acute liver injury is achieved.
FIG. 8 is a CCl provided in example 1 4 And (5) detecting the liver inflammatory factor of the mice with acute liver injury.
FIG. 9 is a CCl provided in example 1 4 And (3) the expression detection result of the mouse liver miR-122 in the acute liver injury model group.
Fig. 10 is an acute liver injury liver eye change caused by APAP provided in example 1.
Fig. 11 is a graph showing that APAP provided in example 1 causes a pathological change in liver due to acute liver injury (HE staining, ×400).
FIG. 12 shows the serum biochemical index test of APAP-induced acute liver injury mice provided in example 1.
FIG. 13 is a graph showing the results of detecting liver antioxidant capacity index of mice suffering from acute liver injury caused by APAP as provided in example 1.
Fig. 14 shows the results of detection of liver inflammatory factors in mice with acute liver injury caused by APAP provided in example 1.
FIG. 15 shows the expression level of liver miR-122 in mice suffering from acute liver injury due to APAP provided in example 1.
FIG. 16 shows the change in liver profile of Alcohol-induced acute liver injury provided in example 1.
FIG. 17 shows the pathological changes in liver pathology of acute liver injury caused by Alcohol provided in example 1 (HE staining,. Times.400).
FIG. 18 shows the results of serum biochemical markers of mice with acute liver injury caused by Alcohol provided in example 1.
FIG. 19 is the liver antioxidant capacity of Alcohol-induced acute liver injury provided in example 1.
FIG. 20 shows the results of liver inflammatory factor detection in mice with acute liver injury caused by Alcohol provided in example 1.
FIG. 21 is a graph showing the results of detection of liver miR-122 expression of an Alcohol-induced acute liver injury mouse provided in example 1.
FIG. 22 shows the effect of the decoction of the composition provided in example 2 on glutamic pyruvic transaminase and glutamic oxaloacetic transaminase of young yaks with liver injury of praziquantel.
Detailed Description
The present invention will be described in detail with reference to the following examples and drawings, but it should be understood that the examples and drawings are only for illustrative purposes and are not intended to limit the scope of the present invention in any way. All reasonable variations and combinations that are included within the scope of the inventive concept fall within the scope of the present invention.
The test methods used in the following examples included:
1. method for evaluating antioxidant effect in vitro:
(1) Total oxidation resistance of Fe
Taking 10 mu L of 1g/mL of the composition decoction, adding 20 mu L of phosphate buffer solution with pH of 6.6 and 20 mu L of 1% potassium ferricyanide solution, mixing, standing at 50 ℃ for 20min, adding 250 mu L of 10% trichloroacetic acid solution, mixing, taking 25 mu L of mixed solution, adding 25 mu L of distilled water and 25 mu L of 0.1% ferric chloride solution, uniformly mixing, standing for 10min, measuring optical density OD at 700nm, and taking a solvent instead of the composition decoction as a blank control.
The total oxidation resistance of Fe is expressed as Δod=od sample-OD blank.
(2) Radical scavenging rate of hydroxyl groups
Sequentially adding 0.5mL of 2mmol/L ferrous sulfate solution and 0.5mL of 6mmol/L hydrogen peroxide into a test tube, shaking uniformly, adding 1.5mL of 6mmol/L salicylic acid, taking out in a water bath at 37 ℃ for 15min, measuring the optical density OD, adding 0.1mL of composition decoction into a blank group, shaking uniformly, continuously heating in the water bath for 15min, taking out 200 mu L of optical density OD sample, and obtaining the hydroxyl radical clearance rate according to the following calculation formula:
clearance (%) = (OD blank-OD sample)/OD blank×100%
(3) DPPH clearance rate
DPPH is prepared into a solution of 0.5mg/mL, and the solution is preserved in dark at 4 ℃ for 3.5 hours, so that the test is finished.
1g/mL of the composition decoction was added to the wells of the ELISA plate at 100. Mu.L/well, and 100. Mu.L of DPPH solution was added thereto to set 3 replicate wells. The blank and background groups were each replaced with 100 μl of purified water, absolute ethanol, and 3 replicate wells were similarly set. And standing for 30min at 37 ℃ in dark condition, and measuring the OD value at 517nm of the enzyme label instrument.
DPPH clearance was obtained as follows:
DPPH clearance (%) = [ OD blank- (OD sample-OD background) ]/OD blank×100%
2. Anti-inflammatory test method:
spectrophotometry is used to determine the inhibition of 5-lipoxygenase activity to characterize the in vitro anti-inflammatory activity of the decoction composition, wherein:
preparation of the substrate: tween-20 was dispersed in 0.02mol/L, PH 9.0.0 borate buffer, a proper amount of linoleic acid was added during shaking, and then L mol/L sodium hydroxide solution was added dropwise to make it clear and transparent and the pH was maintained at 9.0.
Adding 1 mu L of the composition decoction with the concentration of 1g/mL into a 1.5mL EP tube containing 20 mu L of lipoxygenase solution, heating in a water bath at 30 ℃ for 30min, adding 150mL of substrate, putting into a water bath at 30 ℃ again for reaction for 3min, adding 500 mu L of absolute ethyl alcohol, stopping the reaction, adding 500 mu L of distilled water, uniformly mixing, and marking as OD1; the positive control group was 1 μl of distilled water instead of the composition decoction, designated OD2; the negative control group was labeled OD3 with 20 μl of distilled water instead of lipoxygenase; the blank group uses 1 distilled water to replace the composition decoction, 500 mu L absolute ethyl alcohol is added before adding the substrate to stop the reaction, and the reaction is recorded as OD4; repeating the test for 3 times, taking 200 mu L of the composition decoction respectively, detecting the absorbance OD value of each reaction solution at 234nm,
the anti-inflammatory rate was obtained as follows:
anti-inflammatory ratio (%) = [1- (OD 1-OD 3)/(OD 2-OD 4) ] ×100%
3. The bacteriostasis test method comprises the following steps:
the oxford cup bacteriostasis method is adopted for carrying out: after escherichia coli ATCC25922 and staphylococcus aureus ATCC43300 bacterial solutions are diluted according to a certain proportion, a coater is uniformly coated on an MH agar medium, an oxford cup is placed in the MH agar medium, 200 mu L of the composition decoction is taken and added into the oxford cup, and after constant-temperature culture at 37 ℃, the size of a bacteriostasis zone is measured.
4. Composition decoction component analysis:
rutin, luteolin, isochlorogenic acid A and berberine hydrochloride are used as reference components, and a qualitative and quantitative analysis method of the main component of the composition decoction based on HPLC and the content thereof is established to analyze the material basis of the pharmacological effects of the composition decoction. The specific process comprises the following steps:
and accurately weighing appropriate amounts of rutin, luteolin, isochlorogenic acid A and berberine hydrochloride standard substances, preparing mother liquor for standby by chromatographic grade methanol respectively, diluting to appropriate concentration before use, and filtering by a 0.22 μm microporous filter membrane. The chromatographic conditions were as follows: agilent SB-C18 column (4.6mm.times.250 mm,1.5 μm), mobile phase A was acetonitrile, B was 0.2% aqueous phosphoric acid, gradient elution: 0-11 min, 16% -18% of phase A; 11-25min, 18-25% of phase A and 25-30 min; 25% -30% of phase A; 30-40 min, 30-35% of phase A; 40-50 min, 35-40% of phase A; 50-55 min, 40-16% of phase A; the flow rate is 1mL/min, the detection wavelength is 348nm, the sample injection amount is 10 mu L, and the column temperature is 30 ℃.
5. Acute toxicity and cumulative toxicity test:
42 female BALB/c mice were randomly divided into 7 groups, namely, a Control group (Control) and a A, B, C, D, E, F group, and the Control group was filled with physiological saline, and A, B, C, D, E, F groups were filled with the stomach according to the crude drug concentrations of 30 g/kg.bw, 20 g/kg.bw, 13.3 g/kg.bw, 8.9 g/kg.bw, 5.9 g/kg.bw, and 3.9 g/kg.bw, respectively, and observed for 24 hours. If no death or other abnormal phenomenon is detected, the toxicity test is continued for 21 days, and dissection is performed on 22 days to observe the general state, organ index and pathological changes of organs such as heart, liver, spleen, lung, kidney and other solid organs.
6. Evaluation test for acute liver injury protection:
113 male BALB/c mice (18-22 g) were randomly selected as blank groups, and the remaining 105 mice were divided into CCl4 (carbon tetrachloride) group, APAP (acetaminophen) group, and Alcohol (Alcohol) group, and each group was further randomly divided into 5 groups, namely Model group (Model), silybin group (Silybin), high dose group (H), medium dose group (M), and low dose group (L). The blank control group is continuously infused with normal saline for 14 days; the model group does not perform drug lavage; the silybin group is subjected to gastric lavage according to 50mg/kg for 14 continuous days; the high dose group, the medium dose group and the low dose group are respectively and continuously irrigated with stomach according to the crude drug concentration of 10 g/kg.bw, 5 g/kg.bw and 2.5 g/kg.bw for 14 days. Liver injury model replication was performed on day 15, except for the blank, according to the following model replication method.
Chemical acute liver injury model replication (carbon tetrachloride, CCl 4): 10 g/kg.bw of intraperitoneal injection of 1% carbon tetrachloride solution, all test mice were sacrificed on day 16 and dissected;
drug-induced acute liver injury model replication (acetaminophen, APAP): 200 mg/kg.bw of the intraperitoneal injection of acetaminophen solution, all test mice were sacrificed on day 16 and dissected;
model replication of alcoholic acute liver injury (Alcohol): 10 ml/kg.d of distilled spirit with 53 degrees of alcoholic strength of stomach is continuously infused for 5 days, all test mice are sacrificed on day 20 and subjected to sectioning.
Wherein, the evaluation index of acute liver injury is as follows
(1) Index of viscera
The heart, liver, spleen, lung and kidney of the mice are washed clean by sterilized normal saline, and the mice are weighed and the organ indexes are calculated after the filter paper is sucked dry: organ index = organ/body weight x 100%
(2) Histopathological observation of liver
The middle part of the left large leaf of the liver was taken and fixed in 4% paraformaldehyde for 48h, then paraffin-embedded, sectioned, dewaxed, hematoxylin-eosin (HE) stained and finally observed for pathological morphological changes under a microscope.
(3) Serum biochemical index detection
Serum TG, T-CHO content levels were determined according to commercial serum biochemical kit instructions.
(4) Liver antioxidant capacity evaluation and related gene expression level analysis
After preparation of liver homogenates, T-AOC, GSH-Px, SOD, MDA assays were performed according to commercial kit instructions.
Weighing 50mg of liver tissue homogenate, adding 1mL of Trizol lysate for RNA extraction, detecting the concentration of RNA by an ultra-trace nucleic acid protein tester, performing reverse transcription on the RNA into cDNA on a gradient PCR (polymerase chain reaction) tester, and measuring the relative expression amounts of inflammatory factors IL-1 beta, IL-6, TNF-alpha and liver micro ribonucleic acid miR-122 by adopting a real-time fluorescent quantitative PCR method. GAPDH and U6 were selected as reference genes, and the primer sequences are shown in Table 1.
TABLE 1 primer sequences for qPCR detection
7. Effect test on praziquantel induced liver injury in young yaks:
21 healthy young yaks (blank control group, praziquantel group and composition decoction group) were selected for clinical evaluation of the protective effect of the composition decoction on the damage of the young yaks liver caused by excessive praziquantel use. Except for the blank group, praziquantel is orally taken by the other 14 young yaks according to the dosage of 50 mg/kg.bw for 14 days, and the composition decoction group is simultaneously drenched according to the dosage of 10 g/kg.bw. And on the 15 th day, the jugular vein blood is sampled to measure the content of glutamic pyruvic transaminase and glutamic oxaloacetic transaminase in serum, and the appearance, physiological response and the like of young yaks of all groups are closely observed during the test.
The statistics and analysis of the test results are as follows:
statistical data processing using SPSS software, test results toAnd (3) representing. Statistical analysis between groups using LSD test, P<The difference of 0.05 is statistically significant and is marked as a; p (P)<0.01, noted as x; p (P)<0.001, noted as x.
Example 1
A plurality of groups of decoction are prepared according to the following process:
s1: mixing the composition components with the water in a solid-to-liquid ratio of 1:10, soaking for 30min, and decocting for 2 times for 45min each time;
s2: after filtration, the filtrate was collected, and the filtrate was concentrated to 1g/mL (a part of the concentrate was sterilized by filtration through a 0.22 μm needle-type disposable filter) to obtain a decoction.
Wherein the compositions in the different group decoctions satisfy the following components and proportions (table 2):
TABLE 2 orthogonal design level factor table
The in vitro antioxidation, anti-inflammatory and bacteriostasis tests of the different groups of the pharmaceutical compositions are carried out. In the oxidation resistance test, the total Fe oxidation resistance, the hydroxyl radical clearance (%) and the DPPH clearance (%) of the loosestrife are respectively 3.67+/-0.02, 61.07 +/-7.40 and 90.98 +/-1.45, the total Fe oxidation resistance, the hydroxyl radical clearance (%) and the DPPH clearance (%) of the Hangzhou chrysanthemum are respectively 1.17+/-0.02, 66.84 +/-2.46 and 70.77 +/-20.84, and the total Fe oxidation resistance, the hydroxyl radical clearance (%) and the DPPH clearance (%) of the thalictrum hemipterum are respectively 2.41+/-0.32, 48.77 +/-0.76 and 69.85+/-5.29; in the anti-inflammatory test, the anti-inflammatory rates (%) of the loosestrife, the chrysanthemum and the thalictrum hemipterum hance are 69.82 +/-0.46, 78.54+/-8.94 and 64.71 +/-5.84 respectively; in the antibacterial test, the antibacterial circle sizes of the herba Phyllostachydis Henonis to ATCC25922 and ATCC43300 are 14.1+/-0.2 mm and 13.7+/-0.4 mm respectively, the antibacterial circle sizes of the flos Chrysanthemi to ATCC25922 and ATCC43300 are 12.3+/-0.3 mm and 11.6+/-0.5 mm respectively, and the antibacterial circle sizes of the radix et rhizoma Thalictri-pteris to ATCC25922 and ATCC43300 are 13.5+/-1.1 and 12.3+/-0.3 mm respectively.
As can be seen from Table 3, the influence of 3 factors in the antioxidant test was ranked from large to small as B > A > C, and the composition decoction composition with the dominant antioxidant effect was derived as A1B1C2, and was designated as ZJD1.
TABLE 3 oxidation resistance dominated by limit analysis and composite score
| A | B | C | Comprehensive scoring | |
| 1 | 1 | 1 | 1 | 24 |
| 2 | 1 | 2 | 2 | 19 |
| 3 | 1 | 3 | 3 | 17 |
| 4 | 2 | 1 | 2 | 18 |
| 5 | 2 | 2 | 1 | 14 |
| 6 | 2 | 3 | 3 | 23 |
| 7 | 3 | 1 | 2 | 22 |
| 8 | 3 | 2 | 1 | 19 |
| 9 | 3 | 3 | 3 | 17 |
| K1 | 60 | 64 | 57 | |
| K2 | 55 | 52 | 59 | |
| K3 | 58 | 57 | 57 | |
| k1 | 20 | 21.33 | 19 | |
| k2 | 18.33 | 17.33 | 19.67 | |
| k3 | 19.33 | 19 | 19 | |
| R | 1.67 | 4 | 0.67 |
From Table 4, it is clear that the effects of 3 factors in the anti-inflammatory test are ranked as A > B > C from large to small, and the composition decoction composition with inflammation as the dominant component is deduced to be A 1 B 1 C 2 Is denoted as ZJD 1 (same as the drug combinations confirmed by the antioxidation-dominant comparison, they are all denoted as ZJD 1 )。
TABLE 4 anti-inflammatory dominant limit analysis and composite score
As can be seen from Table 5, the influence of 3 factors in the bacteriostasis test is ranked from large to small as A > C > B, and the composition decoction composition with dominant bacteriostasis is deduced to be A 3 B 3 C 1 Is denoted as ZJD 2 。
TABLE 5 analysis of dominant range of antibacterial effect and comprehensive score
| A | B | C | Antibacterial comprehensive score | |
| 1 | 1 | 1 | 1 | 9 |
| 2 | 1 | 2 | 2 | 6 |
| 3 | 1 | 3 | 3 | 6 |
| 4 | 2 | 1 | 2 | 4 |
| 5 | 2 | 2 | 1 | 4 |
| 6 | 2 | 3 | 3 | 4 |
| 7 | 3 | 1 | 2 | 5 |
| 8 | 3 | 2 | 1 | 8 |
| 9 | 3 | 3 | 3 | 9 |
| K1 | 21.0 | 18.0 | 21.0 | |
| K2 | 14.0 | 18.0 | 15.0 | |
| K3 | 22.0 | 19.0 | 19.0 | |
| k1 | 7.0 | 6.0 | 7.0 | |
| k2 | 4.7 | 6.0 | 5.0 | |
| k3 | 7.3 | 6.3 | 6.3 | |
| R | 2.7 | 0.3 | 2.0 |
Finally, the composition decoction composition with highest antioxidant, anti-inflammatory and antibacterial comprehensive activity is compared by taking the antioxidant, anti-inflammatory and antibacterial comprehensive activity as the dominant, the composition decoction composition with highest antioxidant, anti-inflammatory and antibacterial comprehensive activity score is shown in a table 6, and the optimal composition of the composition decoction is finally determined by ZJD 3.
TABLE 6 extremely poor analysis and comprehensive score leading to antioxidant, anti-inflammatory and bacteriostatic comprehensive Activity
Three replicates of composition decoction formulation ZJD3 (A1B 1C 3) were performed separately to verify repeatability, see table 7.
TABLE 7 in vitro Activity repeatability evaluation test of decoction of the composition
Further, the main components and the content of the composition decoction were analyzed based on HPLC (high performance liquid chromatography), wherein the linear regression equation of the mixed standard is shown in table 8, and the liquid chromatograms of the standard and the composition decoction are shown in fig. 1 and 2, respectively.
According to the linear regression analysis, each 1g of the composition decoction contains 0.27mg of rutin, 2.72mg of luteolin, 0.26mg of isochlorogenic acid A and 0.28mg of berberine hydrochloride.
TABLE 8 Standard curve
| Composition of the components | Standard curve equation | R 2 |
| Rutin | y＝12714x–15087 | 0.9999 |
| Luteolin glycoside | y＝32622x–491838 | 0.9927 |
| Isochlorogenic acid A | y＝16182x+113622 | 0.9983 |
| Berberine hydrochloride | y＝51605x-129315 | 0.9964 |
Furthermore, the composition decoction is subjected to channel tropism analysis by a traditional Chinese medicine pharmacological experiment method
The analysis result of the Chinese angelica shows that the rate of the Chinese medicinal decoction entering liver channel reaches 100%, which shows that the Chinese medicinal decoction has the effects of preventing and treating liver diseases, and is shown in Table 9.
Table 9 analysis of the composition decoction for menstruation
Further, the acute toxicity and accumulated toxicity of the composition decoction were tested and evaluated, wherein the administration of the composition decoction showed no significant difference in organ indexes such as heart, liver, spleen, lung, kidney, etc., from the blank control group, and the obtained results are shown in table 10 and fig. 3, which indicate that the composition decoction is safe and nontoxic.
TABLE 10 toxicity test organ index
Further, the decoction of the composition is composed of CCl 4 The influence of the acute liver injury is studied to study the preliminary evaluation of the protective effect of the composition decoction on the acute liver injury.
(1) Organ index:
compared with the blank control group, the intraperitoneal injection CCl 4 After model replication, liver index was reduced, and the composition was improved after decoction treatment, and the liver index of silybin group was similar to that of the blank control group, and there was significant difference from the model group (see table 11).
TABLE 11 CCl 4 Organ index of mice with acute liver injury caused by the method
As can be seen in Table 11, a, there was a significant difference compared to the blank; and b, comparing the model group with the model group, wherein a significant difference exists.
(2) Liver pathological changes
CCl 4 The acute liver injury model group is examined by sectioning, and the liver has red bleeding points and has darker color; the silybin group, the medium dose group and the low dose group show different degrees of white necrosis, and the high dose group has smooth surface and no obvious appearance change, as shown in figure 4.
The liver rope structure of the blank control group is clear, and the liver cells are orderly arranged. In the model group, liver is disturbed, liver Dou Bianzhai and peripheral large-area liver fibrosis appears, liver cells are obviously denatured and necrotized, and interstitial hyperemia cell nuclei are dissolved. Compared with the model group, the swelling deformation and necrosis of the liver cells of each dose group are obviously reduced, as shown in figure 5.
(3) Serum biochemical marker detection result of CCl 4-induced acute liver injury mice
The serum biochemical index detection result shows that after the composition decoction is continuously infused into the stomach for 14 days, the high-dose groups AST and ALT of the composition decoction are consistent with the blank control group. The composition decoction groups have different degrees of inhibition on TG expression, and the silybin group and the composition decoction high-dose group have maintenance effect on T-CHO.
(4) Antioxidant effect
Compared with a blank control group, the liver tissue SOD, GSH-Px and T-AOC of the mice in the model group are obviously reduced, and the MDA content is obviously increased. Compared with the model group, the silybin group and the composition decoction group have different degrees of improvement on SOD, CAT, GSH-Px and MDA in liver tissues, as shown in figure 7.
(5) Detection result of liver inflammatory factor of CCl 4-induced acute liver injury mice
Compared with the model group, the qPCR detection result shows that each dose group of the composition decoction has certain inhibition effect on the expression of IL-1 beta, IL-6 and TNF-alpha, and has statistical significance, as shown in figure 8.
(6) Mouse liver miR-122 detection result of acute liver injury caused by CCl4
Compared with a blank control group, the expression level of the miR-122 in the model group is obviously increased, and the result shows that the H, M dose group after the decoction of the gastric lavage composition has an effect of improving the abnormal expression of the miR-122, as shown in figure 9.
Further, effect test on composition decoction on acute liver injury BALB/c mice caused by APAP:
(1) Index of viscera
Compared with the model group, the liver indexes of H, M, L dose groups show remarkable differences after the gastric lavage composition decoction, which indicates that different dose groups of the composition decoction have the effect of protecting acute liver injury caused by APAP (Table 12).
TABLE 12 organ index of mice suffering from acute liver injury due to APAP
As can be seen in Table 12, a, there was a significant difference compared to the blank; b, compared with the model group, there is a significant difference
(2) Liver pathology and eye changes
The liver of the silybin group mice is blood stasis in different degrees, no obvious pathological change appears on the liver surface of the mice at H, M doses of the composition decoction, and the mice are normally dark red and soft in texture, as shown in figure 10.
HE staining showed that the liver necrosis area of each dosage group of the composition decoction was significantly reduced after stomach lavage, and the liver tissue degeneration necrosis was reduced, indicating that the dry and dry prognosis of the composition decoction can improve the acute liver injury caused by APAP, see fig. 11.
(3) Serum biochemical marker detection for mice with acute liver injury caused by APAP
The effect of the composition decoction on the replication of the AST, ALT, TG, T-CHO APAP model was reduced to various degrees in each dose group, and the level of the composition decoction was basically recovered to that of the blank group, as shown in FIG. 12.
(4) Acute liver injury mouse liver oxidation resistance caused by APAP
Compared with the model group, after the gastric lavage composition decoction, MDA, SOD, GSH-Px and T-AOC abnormalities caused by APAP model replication are relieved to different degrees, as shown in figure 13.
(5) Detection and detection of liver inflammatory factors of mice with acute liver injury caused by APAP
Compared with the model group, the composition decoction has the inhibition effect on the expression of IL-1 beta, IL-6 and TNF-alpha of acute liver injury caused by APAP in different dosage groups and has statistical significance, as shown in figure 14.
(6) Influence of acute liver injury caused by APAP on expression of mouse liver miR-122
Compared with the blank control group, the miR-122 expression quantity of the model group is obviously increased, and the miR-122 expression of the composition decoction H, M dose group tends to the blank control group, as shown in figure 15.
Further, the effect test of the composition decoction on Alcohol-induced acute liver injury BALB/c mice:
(1) Index of viscera
The liver indexes of the silybin group, the composition decoction H and the medium dose group show significant differences from the model group, and are shown in Table 13.
TABLE 13 organ index of mice with acute liver injury due to Alcohol
As can be seen in Table 13, a, there was a significant difference compared to the blank; and b, comparing the model group with the model group, wherein a significant difference exists.
(2) Observation of pathological changes of liver
Compared with the blank control group, the composition decoction has no obvious appearance change of the liver in the high-dose group, some insignificant white necrosis foci appear in the liver in the medium-dose group, and blood stasis appears at the edge of the liver in the low-dose group, as shown in fig. 16.
Liver of the blank control group is healthy in liver cell state after HE staining, and has no pathological changes such as denaturation, necrosis and the like. The degree of hepatocyte injury of each dose group is reduced after the decoction of the gastric lavage composition, and the H group is more obviously relieved, as shown in figure 17.
(3) Serum biochemical marker detection result of mice with acute liver injury caused by Alcohol
Compared with the model group, the silybin group and the composition decoction high-dose group have remarkable improvement effect on serum index abnormality AST, ALT, TG, T-CHO and have statistical significance, as shown in figure 18.
(4) Liver antioxidant capacity of mice with acute liver injury caused by Alcohol
Significant differences appear in mice with acute liver injury caused by Alcohol and in blank control groups, and the dosage groups of the composition decoction reverse the abnormalities of MDA, SOD, GSH-Px and T-AOC after APAP model replication to different degrees, as shown in figure 19.
(5) Alcohol acute liver injury mouse liver inflammatory factor detection result
Compared with the model group, the composition decoction has a certain inhibition effect on the expression of IL-1 beta, IL-6 and TNF-alpha of mice with acute liver injury caused by Alcohol at different doses, and has statistical significance, as shown in figure 20.
(6) Liver miR-122 detection result of Alcohol acute liver injury mice
Compared with a blank control group, the expression level of the miR-122 in the model group is obviously increased, and the abnormal expression of the miR-122 in a high-dose group after the gastric lavage composition decoction is obviously improved, as shown in figure 21.
Example 2
Test of the efficacy of ZJD3 (A1B 1C 3) composition decoction on Praziquantel-induced damage to young yak liver:
after 14 days of continuous administration of the ZJD3 (A1B 1C 3) composition decoction of example 1, namely the composition decoction provided by the present invention, the content of glutamate-pyruvate transaminase and glutamate-oxalate transaminase in the serum of young yaks is obtained, as shown in fig. 22 (in the figure, the significant difference compared with the blank control group is shown, the significant difference compared with the praziquantel group is shown in #), wherein the content of glutamate-pyruvate transaminase is shown in fig. (a), the content of glutamate-oxalate transaminase is shown in fig. (B), and the content of glutamate-pyruvate transaminase in the serum of young yaks after administration is obviously lower than that in the praziquantel group. The content difference of glutamic pyruvic transaminase is not obvious compared with the blank control group. In addition, during the test period, the young yaks of 2 praziquantel groups were found to have different degrees of myotonia, but neither the blank group nor the composition decoction group had the phenomenon. The results show that the composition decoction can relieve adverse reactions such as rise of glutamic pyruvic transaminase and glutamic oxaloacetic transaminase of young yaks caused by praziquantel and muscle tremor, and has a protective effect on young yak liver injury caused by praziquantel.
The above examples are only preferred embodiments of the present invention, and the scope of the present invention is not limited to the above examples. All technical schemes belonging to the concept of the invention belong to the protection scope of the invention. It should be noted that modifications and adaptations to the present invention may occur to one skilled in the art without departing from the principles of the present invention and are intended to be within the scope of the present invention.
Claims (10)
1. The veterinary medicine composition for preventing and treating the acute liver injury of the livestock and poultry is characterized by being prepared from the following components in parts by mass: 20-40 parts of loosestrife, 15-20 parts of chrysanthemum morifolium ramat and 25-35 parts of thalictrum hemipterum.
2. A decoction prepared from the veterinary drug composition of claim 1.
3. A decoction prepared from the veterinary drug composition according to claim 2, which contains the following active ingredients: rutin, luteolin, isochlorogenic acid A and berberine hydrochloride.
4. A method of preparing a decoction from a veterinary composition according to claim 2 or claim 3, comprising:
s1, adding sterilized deionized water into the components for soaking, and then decocting for one or more times to obtain decoction;
s2, concentrating the decoction until the concentration of the solid matters is 1-5 g/ml, thereby obtaining the decoction.
5. The method of claim 4, wherein the solids to liquid ratio of the components to the sterilized deionized water is 1:10 to 1:20.
6. The preparation method according to claim 4, wherein the temperature of the decoction is 55-75 ℃ and the time of each decoction is 30-50 min.
7. Use of a veterinary composition according to claim 1 or a decoction prepared from a veterinary composition according to claim 2 or 3 for the preparation of a veterinary medicament for the prevention and treatment of acute liver injury.
8. Use of a veterinary composition according to claim 1 or a decoction prepared from a veterinary composition according to claim 2 or 3 for the preparation of a veterinary medicament for the prevention and treatment of chemical acute liver injury caused by carbon tetrachloride.
9. Use of a veterinary composition according to claim 1 or a decoction prepared from a veterinary composition according to claim 2 or 3 for the preparation of a veterinary medicament for the prevention and treatment of acute hepatic injury caused by acetaminophen.
10. Use of a veterinary composition according to claim 1 or a decoction prepared from a veterinary composition according to claim 2 or 3 for the preparation of a veterinary medicament for the prevention and treatment of alcoholic acute liver injury caused by alcohol.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105168518A (en) * | 2015-09-12 | 2015-12-23 | 朱巧莲 | Traditional Chinese medicine preparation for treating poultry viral liver injury and preparation method of traditional Chinese medicine preparation |
| CN105727069A (en) * | 2016-03-04 | 2016-07-06 | 青岛市市立医院 | Medicinal composition for treating chronic cholecystitis and preparation method for medicinal composition |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105168518A (en) * | 2015-09-12 | 2015-12-23 | 朱巧莲 | Traditional Chinese medicine preparation for treating poultry viral liver injury and preparation method of traditional Chinese medicine preparation |
| CN105727069A (en) * | 2016-03-04 | 2016-07-06 | 青岛市市立医院 | Medicinal composition for treating chronic cholecystitis and preparation method for medicinal composition |
Non-Patent Citations (2)
| Title |
|---|
| 中草药治疗乙型肝炎的研究进展;王永兵, 毛福林, 王强, 朱荃;中国野生植物资源(第05期);第15-16页 * |
| 浅谈中药与蒙药;张忠斌;吴春艳;丁文雅;;中国民族民间医药(第14期);第5-8页 * |
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