CN102988552A - Traditional Chinese medicine compound radix pulsatillae particle for treating chicken colibacillosis and preparation method and application thereof - Google Patents

Traditional Chinese medicine compound radix pulsatillae particle for treating chicken colibacillosis and preparation method and application thereof Download PDF

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Publication number
CN102988552A
CN102988552A CN201210436936XA CN201210436936A CN102988552A CN 102988552 A CN102988552 A CN 102988552A CN 201210436936X A CN201210436936X A CN 201210436936XA CN 201210436936 A CN201210436936 A CN 201210436936A CN 102988552 A CN102988552 A CN 102988552A
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China
Prior art keywords
radix pulsatillae
solution
chinese medicine
cortex
methanol
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郝智慧
钟英杰
王春元
王艳玲
付海宁
庞云露
沈巍
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QINGDAO CONLINENT ANIMAL PHARMACEUTICAL CO Ltd
Qingdao Continent Pharmaceutical Co Ltd
Qingdao Agricultural University
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QINGDAO CONLINENT ANIMAL PHARMACEUTICAL CO Ltd
Qingdao Continent Pharmaceutical Co Ltd
Qingdao Agricultural University
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Publication of CN102988552A publication Critical patent/CN102988552A/en
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Abstract

The invention provides a traditional Chinese medicine compound radix pulsatillae particle for treating chicken colibacillosis. The traditional Chinese medicine compound radix pulsatillae particle is prepared by the following raw traditional Chinese medicines by weight: 300 to 600g of radix pulsatillae, 300g of coptis root, 450g of phellodendron amurense, and 300 to 600g of cortex fraxini. The traditional Chinese medicine compound radix pulsatillae particle is prepared according to a scientific prescription and process, a scientific burden proportion is gained based on the verification of pilot plant experiments, thus the problem that the quality of the traditional Chinese medicine compound radix pulsatillae particle is subjected to large difference due to the deviation of the extract yield can be avoided, the effective components can be concentrated better, and as a result, the absorption rate and the utilization rate are improved.

Description

A kind of Chinese medicine compound Radix Pulsatillae granule for the treatment of chicken colibacillosis and its preparation method and application
Technical field
The invention belongs to field of veterinary, relate to a kind of compound Chinese medicinal preparation for the treatment of chicken colibacillosis, specifically, relate to a kind of Chinese medicine composition that contains the Radix Pulsatillae, Rhizoma Coptidis, Cortex Phellodendri, Cortex Fraxini and its preparation method and application.
Background technology
The dysentery sexually transmitted disease (STD) card of chicken is caused wherein clinically the most generally coli-infection mostly by bacterial infection.Often use clinically spice or the drinking-water such as antibiotic or antimicrobial drug, although can obtain certain effect, this disease is repeatedly outbreak easily, and can not thoroughly eradicate, consequently cause the treatment cost increase and body is produced toxic and side effects, and cause the appearance of several drug resistance bacterial strain.And Chinese veterinarian has formed the unique theoretical system of a cover in clinical practice in several thousand, and to diarrhea due to damp-heat, the diseases such as dysentery pus and blood have good Therapeutic Method.Curing the Radix Pulsatillae Decoction that holy Zhang Zhongjing invented before more than 1,000 years just has good curative effect to the humid dysentery, and herbal toxic effect is little, residual low, can regulate human body immune function, is difficult for producing drug resistance, so more skillful in disease preventing and treating.
The present invention is according to the dialectical theory of Chinese veterinarian, invented a kind of Chinese medicine composition that contains the Radix Pulsatillae, Rhizoma Coptidis, Cortex Phellodendri, Cortex Fraxini, with the treatment chicken colibacillosis, Chinese veterinarian thinks that the disease that escherichia coli cause belongs to the damp and hot heresy injection part of the body cavity below the umbilicus, housing the bladder, kidneys and bowels, cause the mechanism of qi retardance, abnormal ascending-descending of QI and under the diarrhea inducing, controlling should heat clearing and damp drying, and detoxifcation antidiarrheal is main.Therefore the Radix Pulsatillae that enters blood system among the side of the present invention with bitter cold is monarch, heat-clearing and toxic substances removing, eliminating pathogenic heat from blood to cure dysentery.The Rhizoma Coptidis bitter cold, eliminating fire and detoxication, the thick intestinal of dampness is for controlling the dysentery key medicine; The clear damp-heat in lower-JIAO of Cortex Phellodendri, two medicines are the principal drug assistance heat-clearing and toxic substances removing altogether, especially can dampness control dysentery, is ministerial drug altogether.Cortex Fraxini is pained and tremble with fear, and heat-clearing and toxic substances removing and double with the astringent therapy dysentery relieving is adjuvant.Four medicines share, and play altogether heat-clearing and toxic substances removing, the merit of eliminating pathogenic heat from blood to cure dysentery.I cure mainly diarrhea due to damp-heat by the Radix Pulsatillae granule of company's research and development, and the dysentery pus and blood can be used for the control of bacterial diarrhea.
Summary of the invention
The invention provides a kind of Radix Pulsatillae granule and its preparation method and application, the used Radix Pulsatillae granule of the present invention is easy to use, the mechanism of action is clear and definite, toxic and side effects is little, the treatment chicken colibacillosis does not affect human health.
The invention provides a kind of Chinese medicine composition for the treatment of chicken colibacillosis, said composition is prepared from by the raw material of Chinese medicine medicine of following weight proportion: Radix Pulsatillae 300-600g, Rhizoma Coptidis 300g, Cortex Phellodendri 450g, Cortex Fraxini 300-600g.
Preferably the raw material of Chinese medicine medicine by following weight proportion is prepared from: Radix Pulsatillae 450g, Rhizoma Coptidis 300g, Cortex Phellodendri 450g Cortex Fraxini 450g.
Perhaps the raw material of Chinese medicine medicine by following weight proportion is prepared from: Radix Pulsatillae 600g, Rhizoma Coptidis 300g, Cortex Phellodendri 450g, Cortex Fraxini 600g.
Compositions of the present invention is granule.
The preparation method of compositions of the present invention, comprise the Radix Pulsatillae, Rhizoma Coptidis, Cortex Phellodendri, Cortex Fraxini pulverize, extract, concentrated, filter, granulate, the step of dry granule processed.
Wherein, alcohol reflux is adopted in described extraction.Preferred described extraction may further comprise the steps:
(1) Radix Pulsatillae, Rhizoma Coptidis, Cortex Phellodendri, Cortex Fraxini add 10 times of amount 75% alcohol reflux three times, and each 2 hours,
It is (2) concentrated to extracting solution behind the Recycled ethanol, concentrated,
(3) concentrated solution is prepared into active constituents of medicine, and further processing obtains compositions.
Described granule processed may further comprise the steps:
Active constituents of medicine adds adjuvant, and mixing adds the ethanol moistening, granulate, and oven dry, granulate is made.
Granule of the present invention, preparation method is as follows:
(1) Radix Pulsatillae, Rhizoma Coptidis, Cortex Phellodendri, Cortex Fraxini add 10 times of amount 75% alcohol reflux three times, and each 2 hours,
It is (2) concentrated to extracting solution behind the Recycled ethanol, concentrated,
(3) concentrated solution is dry, pulverizes, and adds sucrose and dextrin, and both ratios are 2:1, and mixing with 70% ethanol moistening, is granulated, oven dry, and granulate is made the 1000g granule.
The present invention also comprises the detection method of compositions of the present invention, particularly granule, comprising: the thin layer of the Radix Pulsatillae is differentiated, the thin layer discriminating of Rhizoma Coptidis, and the thin layer of Cortex Phellodendri is differentiated, the thin layer discriminating of Cortex Fraxini and the step of assay;
Wherein, the discriminating of the Radix Pulsatillae, method is as follows:
(1) preparation of need testing solution
Get this product 0.25 g, porphyrize adds methanol 25 ml, and ultrasonic (power 250W, frequency 35kHz) 15 minutes filters, and filtrate is as need testing solution.
(2) preparation of reference substance solution
Get pulchinenoside B 4Reference substance adds methanol and makes the reference substance solution that per 1 ml contains 0.5 mg.
(3) preparation of negative control solution
Take by weighing the negative sample 0.25g that does not contain the Radix Pulsatillae, press the preparation of test sample preparation method.
(4) differentiate
Take methylene chloride-methanol-water (6.5: 3.5: 1) as developing solvent, point sample amount 2 μ l develop the color and inspect 10% sulphuric acid ethanol test solution.105 ℃ of colour developings.Inspect under the daylight.
Wherein, the thin layer of Rhizoma Coptidis and Cortex Phellodendri discriminating, method is as follows:
(1) preparation of need testing solution
Get this product 0.25 g, porphyrize adds methanol 25 ml, and ultrasonic (power 250W, frequency 35kHz) 15 minutes filters, and filtrate is as need testing solution.
(2) preparation of reference substance solution
Get the berberine hydrochloride reference substance, add methanol and make the reference substance solution that every 1ml contains 0.2 mg.
(3) preparation of negative control solution
Take by weighing jack to jack adapter control sample 0.25g, press the preparation of test sample preparation method.
(4) differentiate
1. developing solvent
A methylene chloride-methanol-water (6.5: 3.5: 1)
2. lamellae
The efficient plate of Merck silica gel G (specification 5 * 5cm)
3. the airtight chromatography cylinder of expansion mode launches before presaturation 20 minutes, ascending development 4cm, and point sample amount 1 μ l,
4. develop the color and dry with inspecting.Put under the ultra-violet lamp (365 nm) and inspect.
Wherein, the discriminating of the thin layer of Cortex Fraxini, method is as follows:
(1) preparation of need testing solution
Get this product 0.1g, porphyrize adds methanol 10 ml, and ultrasonic (power 250W, frequency 35kHz) 15 minutes filters the filtrate evaporate to dryness.Residue adds methanol 2ml makes dissolving, as need testing solution.
(2) preparation of reference substance solution
Get aseculin, aesculetin reference substance, add methanol and make the reference substance solution that every 1ml contains 1mg.Other gets Cortex Fraxini control medicinal material 1g, adds methanol 10ml, and ultrasonic (power 250W, frequency 35kHz) 15 minutes filters, and filtrate is as Cortex Fraxini control medicinal material solution.
(3) preparation of negative control solution
Take by weighing the negative product 0.1g that does not contain Cortex Fraxini, press the preparation of test sample preparation method.
(4) thin layer discrimination test
1. developing solvent
A methylene chloride-methanol-formic acid (6: 1: 0.3)
2. the efficient plate of lamellae Merck silica gel G (specification 5 * 5cm)
3. the airtight chromatography cylinder of expansion mode, ascending development 4cm, point sample amount 1 μ l, temperature and humidity no requirement (NR).
Develop the color and inspect and dry.Put under the ultra-violet lamp (365 nm) and inspect.
Wherein, assay, method is as follows:
Adopt the anemoside B4 in the HPLC method working sample, assay method is as follows: sample is through Dalian Yi Lite Zorbax SB C 18Post (4.6 mm * 200 mm, 5 μ m), mobile phase is acetonitrile-water (26: 74); Ultraviolet detection wavelength: 205nm; Flow velocity: 1ml/min; Column temperature: 30 ℃, get chromatogram, the standard curve under similarity condition calculates the content of anemoside B4 according to chromatogram and anemoside B4,
Regulation assay limit is defined as: by dry product, every 1g this product contains Radix Pulsatillae medical material with anemoside B4 (C 59H 96O 26) meter, must not be less than 10.6mg.
The present invention is granule particularly, prescription and technique preparation according to science sum up the proportion scale of science through the pilot experiment checking, thereby have avoided causing product quality the situation of larger difference to occur because of the deviation of extractum yield, make effective ingredient more concentrated, improved absorbance and utilization rate.Granule of the present invention adopts and extracts, concentrated granulation, both can guarantee taking full advantage of of effective ingredient, has saved natural resources of Chinese medicinal materials, has greatly improved again the bioavailability of drug effect and medicine.Granule of the present invention is fed can adopting to be watered, and is easy to use, is conducive to applying of this medicine.It is respond well that granule of the present invention is used for the treatment of chicken colibacillosis.
Usage and dosage of the present invention is:
Mixed drink: every 1L water, 2 ~ 4g medicine/chicken is used in conjunction 5 days.
Another object of the present invention is the application of pharmaceutical composition of the present invention in the medicine of preparation treatment chicken escherichia coil disease.
Below data by experiment further specify beneficial effect of the present invention.
As medicine group of the present invention, be called Radix Pulsatillae granule with embodiment 1 gained granule
The contrast medicine Radix Pulsatillae is loose for buying gained.
For estimating the clinical drug effect of this product, carried out target animals safety testing, experiment clinical trial and the expansion clinical trial of this product to chicken.
Concrete grammar is as follows:
Because it is a lot of to cause the factor of chicken colibacillosis, in the design process of test grouping, the factors such as weather, environment, sow that should take into full account are on the impact of result of the test, carry out as far as possible under the same conditions thereby guarantee to test, to reduce test error, reach the target of objective evaluation curative effect of medication.
1 target animals safety testing
Select the healthy blue or green foot hens of 120 11 ages in days (public young) to be divided at random 4 groups, 30 every group, be respectively 5 times of Radix Pulsatillae granule recommended dose, 3 times, 1 times administration group and blank group (not administration group), freely mix drink, successive administration 7 days.Observe and measure administration and respectively organized organ index and the histopathology variation of the organs such as the clinical symptoms of chickling, body weight, Average weight increasing a day, average daily feed intake, material anharmonic ratio, hematological indices, Biochemical Indices In Serum and the heart, liver, spleen, stomach, intestinal, fabricius bursa in 14 days.
2 experiment clinical trials
The blue or green foot grass of this test and Selection 18 ages in days male chicken 180 plumages, be divided at random 6 groups, every group of 30 plumages (2 repetitions), except negative control group, all the other each test group chickling all adopt every plumage chest muscle injection to contain the E.coli O78 broth culture 1ml counteracting toxic substances of 109 CFU, set up the chickling model of colibacillosis, 3 test group are pressed respectively high dose (4g/L) behind counteracting toxic substances 12 h, middle dosage (2g/ L), low dosage (1g/ L) gives Radix Pulsatillae particle solution mixed drink, the positive drug matched group gives loose (1.5g/Kgd) suspension crop of the Radix Pulsatillae and gavages, positive controls gives normal saline, successive administration 5 d, observe 10 d, and record sickness rate, mortality rate, survival rate, slaughter the pathological changes situation, gain in weight, the indexs such as feed intake, the effective percentage of each drug test group of statistical analysis, cure rate and material anharmonic ratio are inquired into Radix Pulsatillae granule to the clinical therapeutic efficacy of chicken colibacillosis.
3 enlarge clinical effect trial
Select clinical naturally ill be 2198 of 19~25 age in days Sanhuang chicken of chicken colibacillosis through Laboratory Diagnosed, be divided into 63 of 1106 of Radix Pulsatillae granule therapy groups, 1029 of the loose treatment groups of the Radix Pulsatillae and positive controls.Wherein, Radix Pulsatillae granule therapy group gives the mixed drink of Radix Pulsatillae granule 2.0 g/L, the loose treatment group of the Radix Pulsatillae gives the 1.5g/Kgd mixed feeding by body weight, positive controls gives the mixed drink of normal saline 2.0 ml/L, be used in conjunction 5 days, observed 10 days, and observed and record ill chicken number, death toll, survival number, slaughter the indexs such as pathological changes, average weight gain, the effective percentage of each drug test group of statistical analysis, cure rate and the relative weight gain rate etc. are inquired into Radix Pulsatillae granule to the clinical therapeutic efficacy of chicken colibacillosis.
4 results
4.1 target animals safety testing
The whole experimental observation phase, Radix Pulsatillae granule is tested 1 group (10g/kg body weight), test 2 groups of (6g/kg body weight), test 3 groups of (2g/kg body weight) and negative control group (being untreated), 4 groups of chickens all do not have diseases and death, and spirit, motion, appetite, drinking-water etc. are showed no unusual.Occur dark brown feces but test 1 group of part chickling after 5 days in administration, the second day after administration stops to disappear, and this may be relevant with the color of a large amount of absorption medicines, all the other no abnormality seens.
Hematological indices, Biochemical Indices In Serum and the hepatic and renal function biochemical indicator of measuring in 14 days after 4 groups of chicken administrations be Epidemiological Analysis by statistics, the result shows: Radix Pulsatillae granule is tested 5 multiple dose groups, 3 multiple dose groups and 1 multiple dose group and is compared with negative control group, difference that there are no significant (P〉0.05).
14 days is the off-test load-bearing after administration, cuts open inspection and observes, and each is organized the histopathologies such as Average weight increasing a day, average daily feed intake, material anharmonic ratio and the internal organs heart, liver, spleen, stomach, intestinal, fabricius bursa and changes and the organ index statistical analysis.The result shows: Radix Pulsatillae granule is tested 5 multiple dose groups, 3 multiple dose groups and 1 multiple dose group and is compared with negative control group, Average weight increasing a day, average daily feed intake, material anharmonic ratio and internal organs index there are no significant difference (P〉0.05).The internal organs of each experimental group and matched group chickling are not all found the naked eyes pathological change.
4.2 experiment clinical trial
Give the injection of 18 Japanese instar chickling chest muscles 1 LD 50E.coli O78 broth culture 1ml/ only, can cause and major in 100% morbidity of matched group chickling, and mortality rate reaches 60.0%, and the M ﹠ M of blank group is 0, shows this artificial counteracting toxic substances morbidity modeling success.
Counteracting toxic substances modeling clinical efficacy experimental result shows, the cure rate of Radix Pulsatillae granule high dose group and middle dosage group all not 53.3%, effective percentage is respectively 80.0% and 76.7%, is significantly higher than cure rate 16.7% and effective percentage 56.7%(P<0.05 of the loose medicine matched group of the Radix Pulsatillae) utmost point is significantly higher than cure rate 20.0% and effective percentage 40.0%(P<0.01 of spending matched group together); The cure rate of Radix Pulsatillae granule low dose group is 33.3%, a little more than the cure rate of spending matched group and medicine matched group together (P〉0.05), but the effective percentage of low dose group only is 43.3%, be starkly lower than the effective percentage (P<0.05) of Radix Pulsatillae granule high dose group, middle dosage group and medicine matched group, basically identical with the effective percentage of counteracting toxic substances matched group.The demonstration of growth performance observed result, (4.83 ± 0.49g, 4.73 ± 0.41g and 4.44 ± 0.78g) significantly are lower than blank (6.13 ± 0.15g, P<0.05) to the Average weight increasing a day of Radix Pulsatillae granule high dose group, middle dosage group and medicine matched group.Surface Radix Pulsatillae granule high dose group and middle dosage group are injected the chickling colibacillosis that the artificial counteracting toxic substances of 18 Japanese instar chicklings causes to E.coli O78 broth culture chest muscle all preferably therapeutic effect, and can effectively reduce chicken colibacillosis to the negative effect of growth performance of chicks.Comprehensive the above results, prompting Radix Pulsatillae granule high dose group and middle dosage group solution crop gavage, and successive administration 5d all can effectively treat chicken colibacillosis.
4.3 clinical expanding test result
3 batches of sick chicken treatments are observed merging and are added up, and the loose group of Radix Pulsatillae groups of grains and the Radix Pulsatillae begins spirit improvement, feed intake increase, feces character and color etc. the 3rd day sick chicken of part of administration and progressively takes a turn for the better; The mortality rate for the treatment of phase Radix Pulsatillae groups of grains is 13.7%, the mortality rate of the loose group of the Radix Pulsatillae is 14.3%, the mortality rate of positive controls is 42.9%, analyze through the variance inspection statistics, the mortality rate of the loose group of Radix Pulsatillae groups of grains and the Radix Pulsatillae is without significant difference (P〉0.05), and with the mortality rate of positive controls all in significant difference (P<0.05).The concrete death condition detailed results of respectively organizing every day during the treatment sees Table 1.
Respectively organize the death condition of chicken during table 1 Drug therapy
Annotate: with column data subscript letter different person's significant differences (P<0.05)
3 batches of sick chicken therapeutic tests finish to merge statistics, and the survival chicken number of each treatment group is respectively: 955 of Radix Pulsatillae groups of grains, 882 of the loose groups of the Radix Pulsatillae, 36 of positive controls; Each organizes spirit in all survival chickens, searching for food and drinking water and recover normal chicken number is respectively: 874 of Radix Pulsatillae groups of grains, 811 of the loose groups of the Radix Pulsatillae, 28 of positive controls; Each experimental group is got immediately survival chicken 15 (organizing 5 for every batch) and is slaughtered and cut open inspection, and the chicken number that has no the typical escherichia coli pathological changes such as pericarditis or perihepatitis is respectively: 9 of Radix Pulsatillae groups of grains, 6 of the loose groups of the Radix Pulsatillae, 5 of positive controls.Then the effective percentage of Radix Pulsatillae groups of grains is 79.0%, and the effective percentage of the loose group of the Radix Pulsatillae is 78.8%, all is significantly higher than the effective percentage 44.4%(P of positive controls<0.05); The cure rate of Radix Pulsatillae groups of grains is 51.8%, a little more than the cure rate 34.3% of the loose group of the Radix Pulsatillae, all is significantly higher than the cure rate 19.0%(P of positive controls<0.05).Concrete data and the detailed results of each treatment group see Table 2.
Table 2 is respectively treated the therapeutic outcome of experimental group
Annotate: with column data subscript letter different person's significant differences (P<0.05)
Through 5 days drug treatments and observation in 10 days, each extracts the statistical result showed of weighing of 30 chickens immediately the loose group of second batch off-test Radix Pulsatillae groups of grains and the Radix Pulsatillae, the average weight gain of Radix Pulsatillae groups of grains is 279.6 ± 26.6g, and the average weight gain of the loose group of the Radix Pulsatillae is 267.1 ± 29.3g.36 of 3 batches of positive controls coexistence live chickens, its average weight gain is 272.9 ± 22.9g, statistical check analysis, the average weight gain of the loose group of Radix Pulsatillae groups of grains and the Radix Pulsatillae and the relative weight gain rate be all without significant difference (P〉0.05), and 2 medication groups all are significantly higher than average weight gain and the relative weight gain rate (P<0.05) of positive controls.Detailed results sees Table 3.
Table 3 growth performance measurement result
Annotate: with column data subscript letter different person's significant differences (P<0.05)
Radix Pulsatillae granule is used in conjunction administration in 5 days by the mixed drink of 2.0g/L, can effectively treat chicken colibacillosis, the effective percentage of its effective percentage and the loose 1.5g/kgd mixed feeding of the Radix Pulsatillae and promote that the recovery of the long performance of chicken all living creatures is consistent, and cure rate is better than the Radix Pulsatillae and falls apart.
Detection method of the present invention obtains through screening, and screening process is as follows:
Thin layer is differentiated
The discriminating of 1 Radix Pulsatillae
With pulchinenoside B 4(lot number is: 111766-200601) be the Radix Pulsatillae in reference substance discriminating this product.
Principle: main effective ingredient is saponin component, pulchinenoside B 4Be one of its main effective ingredient, its content is also higher.Triterpene saponin polarity is stronger, and water soluble is soluble in hot water, rare alcohol, hot methanol, hot ethanol, and is several insoluble or be insoluble in acetone, organic solvent that the ether isopolarity is little.The color reaction of triterpenoid compound is more, can be used for the physics and chemistry inspection and knows.
(1) this product 0.25 g is got in the preparation of need testing solution, and porphyrize adds methanol 25 ml, and ultrasonic (power 250W, frequency 35kHz) 15 minutes filters, and filtrate is as need testing solution.
(2) pulchinenoside B is got in the preparation of reference substance solution 4Reference substance adds methanol and makes the reference substance solution that per 1 ml contains 0.5 mg.
(3) preparation of negative control solution takes by weighing the negative sample 0.25g that does not contain the Radix Pulsatillae, presses the preparation of test sample preparation method.
(4) selection of unfolding condition
1. developing solvent
A n-butyl alcohol-Acetic Acid-Water (4: 1: 2)
B methylene chloride-methanol-water (6.5: 3.5: 1)
C toluene-ethyl acetate-acetic acid (20: 10: 0.5)
2. lamellae
The efficient plate of Merck silica gel G (specification 5 * 5cm)
3. the airtight chromatography cylinder of expansion mode, microsyringe (specification: 5.0ul) launch before presaturation 20 minutes, ascending development 4cm, point sample amount 2 μ l, temperature and humidity no requirement (NR).
4. develop the color and inspect 10% sulphuric acid ethanol test solution.105 ℃ of colour developings.Inspect under the daylight.
Grope proof through experiment, select developing solvent condition A and C, the separating degree of need testing solution is poor, and developing solvent condition B, need testing solution with the reference substance solution relevant position on, the speckle of aobvious same color, and the noiseless (see figure 1) of negative solution are therefore adopt this chromatographic condition to differentiate Radix Pulsatillae medical material in the Radix Pulsatillae granule.
(5) brief summary
Physicochemical property according to the monarch drug Radix Pulsatillae in the Radix Pulsatillae granule is groped experimental condition, with pulchinenoside B 4Product take methylene chloride-methanol-water (6.5: 3.5: 1) as developing solvent, have been formulated the thin layer of Radix Pulsatillae medical material and have been differentiated in contrast.
The thin layer of 2 Rhizoma Coptidis and Cortex Phellodendri is differentiated
Take berberine hydrochloride (lot number as: 110713-200609) differentiate Rhizoma Coptidis and Cortex Phellodendri in this product as reference substance.
Principle: the main component in Rhizoma Coptidis and the Cortex Phellodendri is berberine hydrochloride, belongs to isoquinoline alkaloid, and berberine hydrochloride is water insoluble, and can be dissolved in chloroform, and free berberine is soluble in hot water and hot ethanol, is insoluble in benzene, chloroform, acetone.Berberine hydrochloride is aobvious yellow-green fluorescence under 365nm.
(1) preparation of need testing solution
Get this product 0.25 g, porphyrize adds methanol 25 ml, and ultrasonic (power 250W, frequency 35kHz) 15 minutes filters, and filtrate is as need testing solution.
(2) preparation of reference substance solution
Get the berberine hydrochloride reference substance, add methanol and make the reference substance solution that every 1ml contains 0.2 mg.
(3) preparation of negative control solution
Take by weighing jack to jack adapter control sample 0.25g, press the preparation of test sample preparation method.
(4) thin layer discrimination test
With reference to two ones of " Chinese veterinary pharmacopoeia " versions in 2005.
1. developing solvent
A methylene chloride-methanol-water (6.5: 3.5: 1)
B benzene-ethyl acetate-isopropyl alcohol-methanol-water (6: 3: 1.5: 1.5: 0.3)
2. lamellae
The efficient plate of Merck silica gel G (specification 5 * 5cm)
3. the airtight chromatography cylinder of expansion mode launches before presaturation 20 minutes, ascending development 4cm, point sample amount 1 μ l, temperature and humidity no requirement (NR).
4. develop the color and dry with inspecting.Put under the ultra-violet lamp (365 nm) and inspect.
When adopting benzene-ethyl acetate-isopropyl alcohol-methanol-water development system, the blue-fluorescence speckle has interference in the need testing solution, and selection developing solvent condition A, need testing solution with the reference substance solution relevant position on, the fluorescence speckle of aobvious same color, and the noiseless (see figure 2) of negative solution, therefore adopt this chromatographic condition of methylene chloride-methanol-water (6.5: 3.5: 1) in order to differentiate Rhizoma Coptidis and the Cortex Phellodendri in the Radix Pulsatillae granule.
(5) brief summary
According to document material and pharmacopeia primary standard, in the quality standard of Radix Pulsatillae granule, the thin layer of the main component berberine hydrochloride of Rhizoma Coptidis, Cortex Phellodendri differentiated to be optimized and to improve, make identification simple to operate, credible result.
The thin layer of 3 Cortex Fraxini medical materials is differentiated
Take aseculin (lot number as: 110740-200104), aesculetin (lot number as: 110741-200506) and the Cortex Fraxini control medicinal material differentiate Cortex Fraxini in this product as reference substance.
Principle: contain the Coumarins compositions such as aseculin, aesculetin in the Cortex Fraxini, belong to simple Coumarins.The Coumarins composition refers to that a class has the general name of the native compound of benzene a pair of horses going side by side α-pyrone parent nucleus.Structurally can regard the lactone compound that forms along the coumarinic acid dehydration as.The Coumarins composition is blueness or purple fluorescence more, and the umbelliferone class has stronger blue-fluorescence, adds that fluorescence strengthens behind the alkali, color turns green; The aobvious blueness of furocoumarin class or brown.
(1) preparation of need testing solution
Get this product 0.1g, porphyrize adds methanol 10 ml, and ultrasonic (power 250W, frequency 35kHz) 15 minutes filters the filtrate evaporate to dryness.Residue adds methanol 2ml makes dissolving, as need testing solution.
(2) preparation of reference substance solution
Get aseculin, aesculetin reference substance, add methanol and make the reference substance solution that every 1ml contains 1mg.Other gets Cortex Fraxini control medicinal material 1g, adds methanol 10ml, and ultrasonic (power 250W, frequency 35kHz) 15 minutes filters, and filtrate is as Cortex Fraxini control medicinal material solution.
(3) preparation of negative control solution
Take by weighing the negative product 0.1g that does not contain Cortex Fraxini, press the preparation of test sample preparation method.
(4) thin layer discrimination test
With reference to two ones of " Chinese veterinary pharmacopoeia " versions in 2005, the thin-layer identification method operation under the Cortex Fraxini item.
1. developing solvent
A methylene chloride-methanol-formic acid (6: 1: 0.3)
B ethyl acetate-methanol-formic acid (3: 1: 1)
C ethyl acetate-butanone-formic acid-water (5: 3: 1: 1)
2. the efficient plate of lamellae Merck silica gel G (specification 5 * 5cm)
3. the airtight chromatography cylinder of expansion mode, ascending development 4cm, point sample amount 1 μ l, temperature and humidity no requirement (NR).
Develop the color and inspect and dry.Put under the ultra-violet lamp (365 nm) and inspect.
Because content of Aesculetin is lower in the need testing solution, when selecting chromatographic condition, acquire a certain degree of difficulty.Other developing solvent can not well separate aseculin and aesculetin simultaneously.And use developing solvent condition A is methylene chloride-methanol-formic acid (6: 1: 0.3), need testing solution with the relevant position of control medicinal material solution, reference substance solution on, the fluorescence speckle of aobvious same color, and the noiseless (see figure 3) of negative solution, therefore adopt this chromatographic condition to differentiate Cortex Fraxini medical material in the Radix Pulsatillae granule.
(5) brief summary
In the quality standard of Radix Pulsatillae granule, with Cortex Fraxini control medicinal material, aseculin, aesculetin simultaneously in contrast product detect, loose of contrast " Chinese veterinary pharmacopoeia " 2010 editions two Radix Pulsatillaes are lower, method is more simple, repeatability is high, original standard is greatly improved and improves.
Assay
Be the content of effective ingredient during better control is write out a prescription, we use pulchinenoside B in the high effective liquid chromatography for measuring Radix Pulsatillae granule 4Content.After the sample treatment at Dalian Yi Lite Zorbax SB C 18Analyze on post (4.6 mm * 200 mm, the 5 μ m) post, mobile phase is acetonitrile-water (26: 74); Ultraviolet detection wavelength: 205nm; Flow velocity: 1ml/min; Column temperature: 30 ℃.The range of linearity of the method anemoside B4 is 0.524~5.24 μ g as a result, and average recovery is that 99.655%, RSD is 1.113.We think that the method can eliminate the interference of complex component in the Chinese medicine preparation, can carry out assay to anemoside B4, and this method specificity is strong, the result accurately, good reproducibility, can be used for the control product quality.Measure three batches of this product, every 1g this product contains Radix Pulsatillae medical material by anemoside B4 (C 59H 96O 26) meter be respectively 13.44mg, 13.32mg, 13.29mg, average is 13.35 mg.
Content limit should be determined according to surveying containing of the testing result of 3 batch samples and results of stability such as data etc.After measured, this product contains Radix Pulsatillae medical material by anemoside B4 (C 59H 96O 26) meter, every 1g must not be less than 11.67mg.The factors such as increasing, the transfer indfficiency in the preparation process, stability and medical material anemoside B4 content situation of batch inventory when considering large-scale production, assay limit in the draft is defined as: by dry product, every 1g this product contains Radix Pulsatillae medical material with anemoside B4 (C 59H 96O 26) meter, must not be less than 10.6mg.
Description of drawings
The thin layer of Fig. 1 Radix Pulsatillae is differentiated collection of illustrative plates
1: test sample 1(lot number: 2012020101); 2: pulchinenoside B 4Reference substance; 3: test sample 2(lot number: 2012020302); 4: test sample 3(lot number: 2012020503) 5: negative control
The thin layer of Fig. 2 Rhizoma Coptidis, Cortex Phellodendri is differentiated collection of illustrative plates
1: test sample 1(lot number: 2012020101); 2: the berberine hydrochloride reference substance; 3: the Rhizoma Coptidis control medicinal material; 4:(lot number: 2012020302); 5:(lot number: 2012020503) 6: negative control
The thin layer of Fig. 3 Cortex Fraxini is differentiated collection of illustrative plates
1: test sample 1(lot number: 2012020101); 2: aseculin, aesculetin reference substance; 3: the Cortex Fraxini control medicinal material; 4: test sample 2(lot number: 2012020302); 5: test sample 3(lot number: 2012020503) 6: negative control
The specific embodiment
The present invention is further detailed explanation below in conjunction with the specific embodiment, but not as restriction of the present invention.
Embodiment 1
With Rhizoma Coptidis 300g, Radix Pulsatillae 600g, Cortex Fraxini 600g, the Cortex Phellodendri 450g medical material of moisture≤12.0%, add 10 times of amount 75% ethanol, reflux, extract, 3 times, each 2 hours first.Filter, merging filtrate, Recycled ethanol is condensed into thick paste, oven dry, add appropriate amount of auxiliary materials (dextrin: glucose=2:1), mixing, spray is with wetting agent, granulation 1000g, and get final product.
This product is the yellowish-brown granule.Differentiate through thin layer, can record and contain Rhizoma Coptidis, the Radix Pulsatillae, Cortex Fraxini.
Embodiment 2
With Rhizoma Coptidis 100g, Radix Pulsatillae 300g, Cortex Fraxini 300g, the Cortex Phellodendri 250g medical material of moisture≤12.0%, add 10 times of amount 75% ethanol, reflux, extract, 3 times, each 2 hours first.Filter, merging filtrate, Recycled ethanol is condensed into thick paste, oven dry, add appropriate amount of auxiliary materials (dextrin: glucose=2:1), mixing, spray is with wetting agent, granulation 1000g, and get final product.
This product is the yellowish-brown granule.Differentiate through thin layer, can record and contain Rhizoma Coptidis, the Radix Pulsatillae, Cortex Fraxini.
The above only is preferred embodiment of the present invention, is not to be the restriction of the present invention being made other form, and any those skilled in the art may utilize the technology contents of above-mentioned announcement to be changed or be modified as the equivalent embodiment of equivalent variations.But every technical solution of the present invention content that do not break away to any simple modification, equivalent variations and remodeling that above embodiment does, still belongs to the protection domain of technical solution of the present invention according to technical spirit of the present invention.

Claims (10)

1. a Chinese medicine composition for the treatment of chicken colibacillosis is characterized in that, is prepared from by the raw material of Chinese medicine medicine of following weight proportion: Radix Pulsatillae 300-600g, Rhizoma Coptidis 300g, Cortex Phellodendri 450g, Cortex Fraxini 300-600g.
2. a Chinese medicine composition for the treatment of chicken colibacillosis is characterized in that, is prepared from by the raw material of Chinese medicine medicine of following weight proportion: Radix Pulsatillae 450g, Rhizoma Coptidis 300g, Cortex Phellodendri 450g Cortex Fraxini 450g.
3. a Chinese medicine composition for the treatment of chicken colibacillosis is characterized in that, is prepared from by the raw material of Chinese medicine medicine of following weight proportion: Radix Pulsatillae 600g, Rhizoma Coptidis 300g, Cortex Phellodendri 450g, Cortex Fraxini 600g.
4. compositions claimed in claim 1 is granule.
5. the preparation method of compositions claimed in claim 1 is characterized in that, comprise the Radix Pulsatillae, Rhizoma Coptidis, Cortex Phellodendri, Cortex Fraxini pulverize, extract, concentrated, filter, granulate, the step of dry granule processed.
6. preparation method according to claim 5 is characterized in that, described extraction may further comprise the steps:
(1) Radix Pulsatillae, Rhizoma Coptidis, Cortex Phellodendri, Cortex Fraxini are added 10 times of amount 75% alcohol reflux three times, each 2 hours,
It is (2) concentrated to extracting solution behind the Recycled ethanol, concentrated,
(3) concentrated solution is prepared into active constituents of medicine, and further processing obtains compositions.
7. preparation method according to claim 5 is characterized in that, described granule processed may further comprise the steps:
Active constituents of medicine adds adjuvant, and mixing adds the ethanol moistening, granulate, and oven dry, granulate is made.
8. preparation method according to claim 5 is characterized in that, may further comprise the steps:
(1) Radix Pulsatillae, Rhizoma Coptidis, Cortex Phellodendri, Cortex Fraxini are added 10 times of amount 75% alcohol reflux three times, each 2 hours,
It is (2) concentrated to extracting solution behind the Recycled ethanol, concentrated,
(3) concentrated solution is dry, pulverizes, and adds sucrose and dextrin, and both ratios are 2:1, and mixing with 70% ethanol moistening, is granulated, oven dry, and granulate is made the 1000g granule.
9. the detection method of compositions claimed in claim 1 is characterized in that, comprising: the thin layer of the Radix Pulsatillae is differentiated, the thin layer discriminating of Rhizoma Coptidis, and the thin layer of Cortex Phellodendri is differentiated, the thin layer discriminating of Cortex Fraxini and the step of assay;
Wherein, the discriminating of the Radix Pulsatillae, method is as follows:
(1) preparation of need testing solution
Get this product 0.25g, porphyrize adds methanol 25 ml, and ultrasonic 15 minutes, filter, filtrate is as need testing solution;
(2) preparation of reference substance solution
Get pulchinenoside B 4Reference substance adds methanol and makes the reference substance solution that every 1ml contains 0.5mg;
(3) preparation of negative control solution
Take by weighing the negative sample 0.25g that does not contain the Radix Pulsatillae, press the preparation of test sample preparation method;
(4) differentiate
Take methylene chloride-methanol-water (6.5: 3.5: 1) as developing solvent, point sample amount 2 μ l develop the color and inspect 10% sulphuric acid ethanol test solution.105 ℃ of colour developings.Inspect under the daylight;
Wherein, the thin layer of Rhizoma Coptidis and Cortex Phellodendri discriminating, method is as follows:
(1) preparation of need testing solution
Get this product 0.25 g, porphyrize adds methanol 25 ml, and ultrasonic 15 minutes, filter, filtrate is as need testing solution;
(2) preparation of reference substance solution
Get the berberine hydrochloride reference substance, add methanol and make the reference substance solution that every 1ml contains 0.2 mg;
(3) preparation of negative control solution
Take by weighing jack to jack adapter control sample 0.25g, press the preparation of test sample preparation method;
(4) differentiate
1. developing solvent
A methylene chloride-methanol-water (6.5: 3.5: 1)
2. lamellae
The efficient plate of Merck silica gel G
3. the airtight chromatography cylinder of expansion mode launches before presaturation 20 minutes, ascending development 4cm, and point sample amount 1 μ l,
4. develop the color and dry with inspecting.Put under the ultra-violet lamp (365 nm) and inspect;
Wherein, the discriminating of the thin layer of Cortex Fraxini, method is as follows:
(1) preparation of need testing solution
Get this product 0.1g, porphyrize adds methanol 10 ml, ultrasonic 15 minutes, filters the filtrate evaporate to dryness.Residue adds methanol 2ml makes dissolving, as need testing solution;
(2) preparation of reference substance solution
Get aseculin, aesculetin reference substance, add methanol and make the reference substance solution that every 1ml contains 1mg.Other gets Cortex Fraxini control medicinal material 1g, adds methanol 10ml, and ultrasonic 15 minutes, filter, filtrate is as Cortex Fraxini control medicinal material solution;
(3) preparation of negative control solution
Take by weighing the negative product 0.1g that does not contain Cortex Fraxini, press the preparation of test sample preparation method;
(4) thin layer discrimination test
1. developing solvent
A methylene chloride-methanol-formic acid (6: 1: 0.3)
2. the efficient plate of lamellae Merck silica gel G
3. the airtight chromatography cylinder of expansion mode, ascending development 4cm, point sample amount 1 μ l, temperature and humidity no requirement (NR).
Develop the color and inspect and dry.Put under the ultra-violet lamp (365 nm) and inspect;
Wherein, assay, method is as follows:
Adopt the anemoside B4 in the HPLC method working sample, assay method is as follows: sample is through Dalian Yi Lite Zorbax SB C 18Post (4.6 mm * 200 mm, 5 μ m), mobile phase is acetonitrile-water (26: 74); Ultraviolet detection wavelength: 205nm; Flow velocity: 1ml/min; Column temperature: 30 ℃, get chromatogram, the standard curve under similarity condition calculates the content of anemoside B4 according to chromatogram and anemoside B4,
Regulation assay limit is defined as: by dry product, every 1g this product contains Radix Pulsatillae medical material with anemoside B4 (C 59H 96O 26) meter, must not be less than 10.6mg.
10. the application of pharmaceutical composition claimed in claim 1 in the medicine of preparation treatment chicken escherichia coil disease.
CN201210436936XA 2012-11-02 2012-11-02 Traditional Chinese medicine compound radix pulsatillae particle for treating chicken colibacillosis and preparation method and application thereof Pending CN102988552A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104257826A (en) * 2014-09-24 2015-01-07 河南亚卫动物药业有限公司 Traditional Chinese medicine pycnonotus sinensis fermented granule for livestock and poultry and preparation method of traditional Chinese medicine pycnonotus sinensis fermented granule
CN104758407A (en) * 2015-04-22 2015-07-08 天农大(天津)生物科技有限公司 Traditional Chinese medicine composition for treating hemorrhagic enteritis in turkey and preparation method of traditional Chinese medicine composition
CN108152392A (en) * 2017-12-08 2018-06-12 宋瑞 The detection method of angelica dahurica coumarin in a kind of veterinary drug

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CN1939467A (en) * 2006-09-28 2007-04-04 天津生机集团有限公司 Chinese medicine for preventing and treating fowl colibacillosis

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Cited By (3)

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Publication number Priority date Publication date Assignee Title
CN104257826A (en) * 2014-09-24 2015-01-07 河南亚卫动物药业有限公司 Traditional Chinese medicine pycnonotus sinensis fermented granule for livestock and poultry and preparation method of traditional Chinese medicine pycnonotus sinensis fermented granule
CN104758407A (en) * 2015-04-22 2015-07-08 天农大(天津)生物科技有限公司 Traditional Chinese medicine composition for treating hemorrhagic enteritis in turkey and preparation method of traditional Chinese medicine composition
CN108152392A (en) * 2017-12-08 2018-06-12 宋瑞 The detection method of angelica dahurica coumarin in a kind of veterinary drug

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