Summary of the invention
The invention provides a kind of method for preparing and application that prevents and treats the newborn recovering particles of herbal mixture granule stilbene grass of bovine subclinical mastitis.Shortcomings such as the product that has solved present treatment bovine subclinical mastitis is less, is mostly antibiotic, and antibiotic resistance and residue problem are serious.
For solving the problems of the technologies described above, the present invention adopts following technical proposals to be achieved.
A kind of Chinese medicine granules of treatment bovine subclinical mastitis, this granule is prepared from following Chinese medicine raw materials by weight proportion medicine, Radix Astragali 350g~800g, Herba Leonuri 100g~400g, Semen Vaccariae (parched) 100~400g.
Preferably, this granule is prepared from following Chinese medicine raw materials by weight proportion medicine, Radix Astragali 400g, Herba Leonuri 300g, Semen Vaccariae (parched) 300g.
The present invention also provides the method for preparing of Chinese medicine granules of the present invention, this method be by the Radix Astragali, Herba Leonuri and
Semen Vaccariae (parched) adds adjuvant behind concentrate drying behind water extraction, granulate, and promptly gets;
Wherein, water extraction process is:
A: the Radix Astragali, Herba Leonuri and Semen Vaccariae (parched), with the water infiltration of 10 times of weight 30 minutes;
B: heating and refluxing extraction is three times then, each 1h, and each water of all using 10 times of weight is carried
Get the extracting solution that the back merges three times, filter.
Behind the extracting liquid filtering, concentrate drying is ground into fine powder, adds adjuvant, and mix homogeneously adds adhesive, granulate, and drying, granulate promptly gets.
Granule of the present invention can be used for the control of bovine subclinical mastitis.
Granule of the present invention is called the newborn recovering particles of stilbene grass, and instructions of taking is following: mix by the following dosage of 750g/ head/d and raise successive administration 7d, and without any side effects to milk cow in lactation period, and can improve the above milk yield of 1.08Kg/ head/d.Clinically, mix by body weight 150g/ head/d and to raise administration, logotype 5d can effectively prevent and treat bovine subclinical mastitis.
The screening process of the inventive method is described below in conjunction with test method.
(1) selection of infiltrating time
Whether get the medical material of 1/5 recipe quantity, add 10 times of water gagings respectively, whenever got wherein each flavor medical material at a distance from 10 minutes, cut off, observe the medical material center and soak into, result of the test is seen table 1.
Table 1 infiltrating time is investigated
The result shows: adopt water infiltration that medical material is soaked into and pass through, so infiltrating time is decided to be 30 minutes.
(2) investigation of extraction process
List of references, the main pharmacodynamics composition of each medical material of these article is a water solublity, so be to extract solvent with water, adopts the heating and refluxing extraction method to extract.In conjunction with experience; The main technologic parameters that influences water extraction comprises extraction time, amount of water, extraction time; Still with extraction time, amount of water, extraction time be the investigation factor; Extracting usual amounts with each factor be the level of each factor, serves as to investigate index with the index components calycosin glucoside of the Radix Astragali in the prescription, preferably the extraction process parameter.Carry out L9 (3
3) orthogonal test, factor design and result of the test are seen table 2~table 4.
Table 2 factor level table
Table 3 Study on extraction L
9(3
3) orthogonal table
Table 4 extraction process range analysis table
Presentation of results is the significance influence factor through variance analysis: A, B factor, A>B>C in three factors.Think that through the extreme difference table analysis optimised process is A
3B
3C
2, promptly adopt to add 12 times of water gagings, extract each 1 hour 4 times.Through range analysis, for A factor, K
A3-K
A1=47.1, K
A3-K
A2=2.8, promptly extract 3 times with extract 4 times less for the influence of this quality, for B factor, K
B3-K
B1=43.7, K
B3-K
B2=1.4, it is less for the influence of this quality to add 12 times of amounts and 10 times of amounts, considers so combine to produce, and reduces energy consumption, practices thrift finished product, reduces and pollutes, and adopts to add 10 times of water gagings extractions 3 times.Composite score and produce and actually think that these article optimum extraction process is: add 10 times of water gagings and soak into after 0.5 hour, extracts 3 times, at every turn 1h.Below, further specify beneficial effect of the present invention through experimental data:
With target animals safety testing and the clinical prevention effect test of the careless newborn recovering particles of the stilbene of embodiment 1 gained to bovine subclinical mastitis.
(1) target animals safety testing
1 materials and methods
1.1 medicine, reagent and instrument
Receive the newborn recovering particles of reagent thing stilbene grass, every bag of 1000g, every 1g contains crude drug 1g, and lot number is 2010012302, is provided by Qingdao KDN Pharmaceutical Co., Ltd..
Reagent biochemical reagents detection kit is all available from Zhongsheng Beikong Biological Science & Technology Co., Ltd.; Other relevant chemical reagent are homemade analytical pure.
Key instrument UL40BC type milk composition automatic analyzer, glamour 2000 automatic clinical chemistry analyzers (U.S. M.D. instrument company, glamour 2000), biological microscope, superclean bench, centrifuge, analytical balance etc.
1.2 experimental animal
The test pasture is Yangzhou University experiment pasture, and kind is Dutch Fresian, in No. 5 cowshed, chooses 30 of healthy cows.
1.3 the test period
Test from April 6th, 2010 to April 21, the test period is 15 days.
1.4 test method
30 of the healthy cows of No. 5 cowshed of selection are divided into 3 groups at random by milch cow livestock on hand numbering and cattle berth, confirm target animals safety testing dosage with reference to " People's Republic of China's veterinary drug allusion quotation " and rat acute toxicity test result.Wherein 2 administration groups are each 10; Promptly test the careless newborn recovering particles of stilbene of 2 groups of 450g/ head/d of 1 group of 750g/ head/d and test by 5 times of the newborn recovering particles clinical treatment test of stilbene grass RD 150g/ head/d with 3 multiple doses respectively; In irritating clothes, successive administration 7d after with an amount of eliminating cold for resuscitation water dissolution between every morning 8 ~ 9.10 of negative control group are not handled, the normal raising.After the administration every day each cow head of observed and recorded spirit, search for food, drink water, motion and milk yield, observe 14d continuously; And 7d, 14d respectively organize milch cow through tail vein blood after administration; Carry out the detection of hemogram regulating liver-QI, renal function: hemogram detects and comprises RBC, white blood count and differential, quantitative hemoglobin etc.; Liver, renal function conventional index are like glutamic oxaloacetic transaminase, GOT (AST or SGOT), glutamate pyruvate transaminase (ALT or SGPT), total protein (TP), serum albumin (Alb), blood glucose (GLu), creatinine (Cr), blood urea nitrogen (BUN), T-CHOL (TCH) and triglyceride (TG) etc.The feeding and management of whole observation period is all undertaken by routine.
1.5 date processing
Data are represented with
, organize the significance test of a data with SPSS 13.0 application softwaries.Adopt the significant difference of each item indexs such as blood biochemical between t check comparative test group and matched group.
2 results
2.1 clinical manifestation
The whole test observation period; The newborn recovering particles of stilbene grass is tested 1 group of (2 groups on 750g/ head/d), test (450g/ head/d) and negative control group (the normal raising of being untreated); Disease and death all do not take place in 3 groups of milch cows, and equal no abnormality seen such as spirit, motion, appetite, drinking-water.
2.2 milk yield
After finishing to 14 day observation period respectively the average daily milk yield result that respectively organizes of statistics show; The average daily milk yield of stilbene newborn recovering particles 5 multiple dose groups of grass and 3 multiple dose groups has all increased more than the 1.08Kg; Apparently higher than negative control group, but there was no significant difference between 3 groups, detailed results is seen table 5.
Average daily milk yield
n=10 is respectively organized in table 5 test
Annotate: difference is remarkable (P>0.05) not
2.3 routine blood test and serum biochemistry parameter
7d, 14d measure after 3 groups of milch cow administrations routine blood test, serum biochemistry index and hepatic and renal function biochemical indicator are through statistical analysis; The result shows: the newborn recovering particles of stilbene grass is tested 5 multiple dose groups and is compared with negative control group with test 3 multiple dose groups; Difference that there are no significant (P>0.05), detailed results is seen table 6, table 7.
7d respectively organizes routine blood test and serum biochemistry mensuration result
n=10 of milch cow after table 6 administration
Annotate: difference is remarkable (P>0.05) not
14d respectively organizes routine blood test and serum biochemistry mensuration result
n=10 of milch cow after table 7 administration
Annotate: difference is remarkable (P>0.05) not
3 interpretations of result
The newborn recovering particles main component of stilbene grass is the Radix Astragali, Herba Leonuri, Semen Vaccariae, and according to People's Republic of China's veterinary drug allusion quotation record, the Radix Astragali, Herba Leonuri, Semen Vaccariae 3 flavor Chinese medicines are used by RD most of domestic animals are had no side effect; So far do not see the report of this 3 flavor Chinese medicine to the milch cow toxic side effect yet.The newborn recovering particles of this stilbene grass is by being higher than 3 times of clinical RD (750g/ heads/d) and 5 times of (dosed administrations of 450g/ head/d); Continuous 7d; To the spirit of milch cow, search for food, move, physical signs such as drinking-water and serum routine biochemistry index all reach the hepatic and renal function biochemical indicator does not have obvious influence, show that the newborn recovering particles of stilbene grass does not also have clinical type toxic and side effects to milch cow.
4 conclusions
The newborn recovering particles of stilbene grass is obeyed to milk cow irrigation according to the dosage of 450g/ head/d and 750g/ head/d, does not cause test cattle morbidity or dead; Spirit, appetite, motion and drinking-water are had no adverse effects; Blood and serum biochemistry index are had no adverse effects; And can improve milk production of cow in right amount.The result of the test prompting, the newborn recovering particles of stilbene grass has higher clinical drug safety property to milch cow.
(2) clinical prevention effect test
1 materials and methods
1.1 medicine, reagent and instrument
Receive the newborn recovering particles of reagent thing stilbene grass, every bag of 1000g, every 1g contains crude drug 1g, and lot number is 2010012302, is provided by Qingdao KDN Pharmaceutical Co., Ltd..
Control drug ten thousand newborn health, commodity are called the mastitis health, every bag of 130g, lot number: 0100116, main component: levamisole hydrochloride, Herba Epimedii, the Radix Astragali, Development Co., Ltd produces by the permanent Ji Lide biotechnology in Tianjin.
Reagent mammitis of cow detectable (HMT) is provided by Zhejiang University's Food Science and fermentation engineering institute, and other conventional reagent are homemade analytical pure level product.
Key instrument UL40BC type milk composition automatic analyzer, mastitis plastics diagnosis dish, medical incubator, biological microscope, superclean bench, centrifuge, analytical balance, steam autoclave sterilizer etc.
1.2 experimental animal
The test pasture is Yangzhou University experiment pasture, and kind is Dutch Fresian.Select to suffer from naturally this lactation period latent mammitis milch cow and healthy cow respectively.Wherein the case diagnosis standard is following:
1.2.1 latent mammitis diagnosis
Detect before milking by the report method.At first, squeeze and go each nipple preceding 3, press right front, right back, left front and left back order with plastics diagnosis dish (the milky plate of dark 1.5cm, diameter 5cm) then milk with warm water towel wiping breast and the nipple of sterilizing; Access the about 2ml milk of each nipple appearance; Add equivalent HMT diagnosticum, horizontal diagnosis dish also makes it to be the concentric circular rotation and shakes, and lets milk appearance fully mix with diagnosticum; Behind 10 ~ 20s, according to table 8 standard determination result.
Mastitis test (HMT) criterion of table 8 Hangzhou
Suspicious milk appearance replication once, as still being the suspicious feminine gender that then is judged to be.Add up for ease; The standard in the positive breast district of detecting HMT "+, ++, +++" use numeral " 1 minute, 2 minutes, 3 minutes " expression latent mammitis inflammation degree respectively, every cattle is got the positive breast district score summation that detects in 4 breast districts is suffered from latent mammitis as this cattle level indexs.
1.2.2 latent mammitis milch cow choice criteria
Milch cow whole body and breast do not have the visible ANOMALOUS VARIATIONS of naked eyes; 4 breast districts (4 nipples) all can extrude milk, and HMT detection reaction grade is being that the scorching card degree of latent mammitis being judged to of (the detection summations in 4 breast districts) more than " 1 minute " suffered from the latent mammitis milch cow more than "+".
1.3 the test period
Whole test from May 14th, 2010 to June 30, the test period is 48 days.
1.4 test method
Because it is a lot of to cause the factor of bovine subclinical mastitis; In the design process that test is divided into groups; Should take into full account of the influence of factors such as weather, environment, milch cow parity, lactogenic cycle to result of the test; Carry out as far as possible under the same conditions thereby guarantee to test,, reach the target of objective evaluation curative effect of medication to reduce test error.Therefore, whole test adopts with house controlled trial principle, makes every effort to reduce the influence to experimental result of main, objective factor.Duration of test, feedstuff and herbage that the test milch cow gives not add any antibiotics are raised by conventional feeding and management.
1.4.1 experimental clinical trial
What selection met bovine subclinical mastitis diagnostic criteria and choice criteria suffers from 75 of latent mammitis milch cows naturally; After confirming that suffering from cattle did not use any Drug therapy in nearest January; Be divided into 5 groups at random by milch cow livestock on hand numbering: i.e. the high, medium and low dose groups of the stilbene newborn recovering particles of grass; Mastitis health medicine contrast (positive drug contrast) group and positive control (ill not administration) group, every group each 15.15 healthy cows of negative control group are normally fed, and do not do any place.The newborn recovering particles test group route of administration of all stilbenes grass is all obeyed in irritating after with an amount of eliminating cold for resuscitation water dissolution between every morning 8 ~ 9, and mastitis Kang Ze mixes concentrate and feeds, and 1 time/d, logotype 5d.Number set up case history archive by cattle,, observe every day also by the mental status, the diet situation of group by head record milch cow from administration same day.0d, 5d, 10d after medication; Detect breast district number and the degree of respectively testing cattle trouble latent mammitis with the HMT diagnosticum; And the breast appearance of gathering 4 newborn districts of each cow head is sent milk processing factory of Yangzhou University milk assay laboratory, fat, lactose, Protein content and the density of employing UL40BC type milk composition automatic analyzer mensuration milk.Statistics is respectively organized the average daily milk yield during cure rate, improvement rate, total effective rate and every group of cattle are treated respectively.Concrete grouping and medicine-feeding test are handled and are seen table 9.
Each test group disposition of table 9
1.4.2 prophylactic tria
Select 90 of the healthy milk cow in lactation period of same cowshed, deposit by the hurdle numbering by milch cow and be divided into 3 groups at random, 30 every group; Wherein prevent 1 group to give stilbene grass newborn recovering particles 150g/ head/d, miscible in putting in the drinking trough between every morning 8 ~ 9 from drink (every cattle has independent drinking trough in the test cattle farm), prevent 2 groups to give positive drug mastitis health 130g/ head/d (by specification dosage); Mix concentrate and feed administration, 1 time/d, logotype 3d; Prevent 3 groups of negative matched groups, normally feed, do not do any place; Observe 30d; 0d, 15d, 30d after medication detect with the HMT diagnosticum and respectively to test breast district number and the degree that cattle suffers from latent mammitis, write down the milk yield of every cattle every day simultaneously.Add up 3 groups of average daily milk yield and healthy cow head number at last respectively and account for the ratio of this group milk ox head number, calculate prevention efficient, judge preventive effect.
1.4.3 therapeutic outcome is judged
With the criterion of HMT diagnosticum testing result as medication effect, therapeutic effect divides recovery from illness, effective, invalid 3 ranks, and concrete standard is following:
The milk appearance in 4 breast districts of the ill milch cow in recovery from illness treatment back are detected through the HMT diagnosticum, and are all negative, and whole body and The entire breast do not have macroscopic pathological phenomenon person, are judged to recovery from illness.
Effectively the milk appearance in 4 breast districts of the ill milch cow in treatment back are detected through the HMT diagnosticum, and its latent mammitis inflammation degree integration descends more than 1.5 minutes (i.e. 1.5 "+"), and whole body and The entire breast do not have macroscopic pathological phenomenon person, are judged to effective.
Though ill milch cow whole body and The entire breast do not have macroscopic pathological phenomenon after the futile treatment, the milk appearance in 4 breast districts is detected through the HMT diagnosticum, and the ill degree integration of its latent mammitis does not descend; Or the person of changing the clinic mastitis into more than 1 breast district, it is invalid all to be judged to.
Recovery from illness, effective number sum are total effective number, and the ratio that accounts for this group treatment number according to total effective number is calculated total effective rate; The ratio that accounts for this group number according to invalid number is calculated inefficiency.Respectively organize the difference of total effective rate with X 2 test.
Preventive effect, the feminine gender milk ox head number that detects through the HMT diagnosticum with 15d during the prophylactic tria and 30d accounts for the ratio of this group milk ox head number and calculates prevention efficient, with X 2 test relatively each group prevent the difference of efficient.
1.4.4 data statistics
Test data carries out statistical disposition with SPSS 13.0 softwares; The result is with average
± standard deviation (SD) expression, and P<0.05 is a significant difference.
2 results
2.1 all test group of clinical efficacy result of the test are through 1 time/d; 5d administration continuously; Detect and the milk composition analysis with the HMT diagnosticum in 5d, 10d; The result shows: the total effective rate of the newborn recovering particles high dose group of stilbene grass, middle dose groups, low dose group and positive drug matched group is respectively: 86.7%, 86.7%, 66.7% and 73.3%; Compare significant difference (P < 0.05) with 0.0% of positive controls, explain that stilbene newborn recovering particles of grass and positive drug mastitis health all have curative effect preferably to bovine subclinical mastitis.The total effective rate of the newborn recovering particles high dose group of further careful relatively stilbene grass, middle dose groups, low dose group and 4 medication groups of positive drug mastitis health; But the total effective rate of stilbene newborn recovering particles high dose group of grass and middle dose groups is 86.7%; Again apparently higher than low dose group and positive drug matched group (P < 0.05), and its cure rate of 40.0% also is significantly higher than the stilbene newborn recovering particles low dose group of grass and positive drug matched group 26.7% (P < 0.05).Relatively the latent mammitis inflammation degree before and after the treatment can be found out; The inflammation degree of all ill cattle is all more than 3.47 minutes before the treatment; And the inflammation degree of the newborn recovering particles high dose group of treatment back stilbene grass, middle dose groups, low dose group and positive drug matched group all dropped to below 2.50 minutes; With 3.80 minutes of positive controls significant difference is arranged, detailed results is seen table 2.
2.2 the variation of milk yield and dairy food quality is observed through 5d administration 10d before and after the treatment; The milk yield of stilbene grass high, medium and low 3 dosage treatment groups of newborn recovering particles and positive drug matched group all has in various degree to be increased; With compare before the treatment, in the newborn recovering particles of stilbene grass the average daily milk yield increase of every cattle of dose groups the most obvious, be 1.6Kg/ head/d; The newborn recovering particles high dose group of stilbene grass is taken second place; Be 1.2Kg/ head/d, compare with the 0.4Kg/ head/d of newborn recovering particles low dose group of stilbene grass and positive drug matched group that there were significant differences (P < 0.05), compared utmost point significant difference (P < 0.01) with 0 increase of positive controls.It is then not obvious that the dairy food quality composition changes, and removes that each medication group of butter oil content treatment back all slightly raises but the difference not remarkable (P>0.05), lactoprotein and lactose content and newborn density have no significant change, and detailed results is seen table 10.
The clinical trial of the newborn recovering particles treatment of table 10 stilbene grass bovine subclinical mastitis is n=15 as a result
Annotate: with line data subscript letter different person's significant differences (P<0.05).
Milk yield and mass component thereof change n=15 before and after table 11 treatment
Annotate: with line data subscript letter different person's significant differences (P<0.05).
2.3 prophylactic tria result
Experimental observation is during to 30d; Prevent 3 groups to be that the newly-increased sickness rate of negative control group reaches 16.7%; To prevent 1 group be the newborn recovering particles group of stilbene grass and 2 groups of preventions are that the newly-increased sickness rate of positive control mastitis health group is respectively 3.3% and 6.7%, significantly is lower than negative control group (P < 0.05), shows that careless newborn recovering particles of stilbene and positive control medicine have clear and definite preventive effect to bovine subclinical mastitis; And can prevent the decline of milk yield, detailed results is seen table 12.
Table 12 prophylactic tria is n=30 as a result
Annotate: with column data subscript letter different person's significant differences (P<0.05).
3 discuss
3.1 the newborn recovering particles 150g/ head/d of stilbene grass, 1 time/d, logotype 3d, the observation through 30d shows, can obviously reduce the newly-increased sickness rate (drop to 6.67% from 16.7%, P < 0.05) of latent mammitis, and can prevent the decline of milk yield.
3.2 the clinical treatment result of the test shows; The cure rate and the total effective rate of stilbene newborn recovering particles high dose group of grass and middle dose groups are the highest; All apparently higher than newborn recovering particles low dose group of stilbene grass and positive drug matched group, wherein there is significant difference (P < 0.05) between the cure rate of careless newborn recovering particles high dose group of stilbene and middle dose groups and low dose group, positive drug matched group and positive controls; Though the newborn recovering particles treatment of stilbene grass back dairy food quality component target does not have significant change at short notice; But the average daily milk yield of the stilbene newborn recovering particles high dose group of grass and middle dose groups every cattle when realizing 40% cure rate has improved 1.6Kg, and with low dose group, positive drug matched group and positive controls significant difference is arranged all.Therefore, dosage was advisable during the dosage of the newborn recovering particles treatment of suggestion stilbene grass bovine subclinical mastitis was selected, i.e. 150g/> head/d, and logotype 5d just can reach therapeutic effect.
4 conclusions
The newborn recovering particles of stilbene grass is pressed 150g/ head/d, and logotype 5d can effectively treat bovine subclinical mastitis, and can improve milk yield gradually.
The newborn recovering particles of stilbene grass is pressed 150g/ head/d, and logotype 3d can effectively prevent the generation of bovine subclinical mastitis.