CN104473872A - Plant polysaccharide oil emulsion and preparation method and application thereof - Google Patents
Plant polysaccharide oil emulsion and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a preparation method of a plant polysaccharide oil emulsion. The method comprises the following steps: 1) evenly mixing span-80 with edible vegetable oil at the volume ratio of (10:90) to (16:84), so as to obtain an oil phase; 2) dissolving plant polysaccharide into a tween-80 water solution with volume concentration of 5%-7%, and adjusting the pH value to be 2-7, so as to obtain the plant polysaccharide water solution containing 8-64mg of plant polysaccharide in each ml as a water phase; and 3) fully mixing the water phase with the oil phase at the volume ratio of 1 to (1-2), and emulsifying, so as to obtain the plant polysaccharide oil emulsion. The plant polysaccharide is astragalus polysaccharide, atractylodes macrocephalaon polysaccharide, polysaccharides from radix codonopsis or glycyrrhiza polysaccharide. The plant polysaccharide oil emulsion can be used as an immunologic stimulant or a treatment medicine for cow mastitis.
Description
Technical field
The present invention relates to a kind of vegetable polysaccharides oil emulsion and its production and use.
Background technology
Polysaccharide is the polymer that a kind of monosaccharide groups by more than 10 is linked together by glycosidic bond, in the body being extensively present in animal and plant and in the cell wall of microorganism.The polysaccharide obtained from Chinese herbal medicine extracting has various biological function.Angelica Polysaccharide is by reducing the Treg cell proportion of tumor-bearing mice and improving spleen lymphocyte proliferation ability and play immunoregulation effect [1].Lycium barbarum polysaccharide can make CD4+T cell proportion in esophageal carcinoma rat peripheral blood raise, and CD4+CD25+Tr cell proportion declines, and IL-10 secretion reduces and the secretion of IL-2 increases, and improves experimental esophageal carcinoma rat cell immunologic function [2].Liuwei Dihuang polysaccharides oral administration can antagonism cyclophosphamide to mouse boosting cell antibody tormation inhibitory action, external application can promote that mouse spleen cell proliferation reacts, and raises splenocyte and produces the level of antibody, increase the number [3] of spleen antibody-producting cell.Porphyra haitanensis polysaccharide and catabolite thereof significantly can promote that T cell is bred, suppress B cell proliferation [4].The ganoderan adding variable concentrations in NK cell can improve NK cytoactive, promotes tumor necrosis factor ɑ, IL-2, interferon gamma release [5].Lycium barbarum polysaccharide can promote to produce multiple lymphocyte factor as macrophage shifter factor, lymphotoxin transfer factor etc.These factors in vivo can the division growth of Promote immunity competent cell, improves the phagocytosis of macrophage and the kill capability [6] of T cell.Herba Cistanches polysaccharides obviously can increase the release [7] of RAW264.7 cell TNF-α and IL-1.Astragalus polysaccharides (APS) [8] can activate Mus peritoneal macrophage induction NO and produce, and induce transcribing of NO synthase (iNOS) by activating transcription factor NF-κ B, play immunological enhancement.Herba Cistanches polysaccharides, by improving macrophage phagocytic and secretory function, activated macrophage, strengthens immunologic function and plays antitumor action [9].Due to the biological function that polysaccharide is excellent, polysaccharides of traditional Chinese medicine is widely applied at field of medicaments, for preventing and treating human diseases.Polysaccharides of traditional Chinese medicine is also widely used in animal farming industry, prevents and treats Animal diseases, reduces the use of chemicals, reduces medicine in animal body residual.
The growing with each passing day to Chinese crude drug demand along with medical industry and animal farming industry, natural resources of Chinese medicinal materials faces immense pressure.According to investigations, the stock number of 100 several kinds of Chinese medicinal materials such as resources of medicinal plant Radix Glycyrrhizae, Glycyrrhiza glabra L., Herba Cistanches, Radix Stellariae, Radix Arnebiae (Radix Lithospermi), the Radix Astragali generally declines, and has influence on the pharmaceutical drugs of more than 60 medical material kind.More than the 30 kind of medical materials such as Radix et Rhizoma Dysosmatis, the Cortex Eucommiae, Radix aconiti hemsleyani, wild mountain ginseng, Herba hedyotis costatae, little Radix Berchemiae Giraldianae, magnolia officinalis rehd.et wils.var.biloba rehd.et wils., because of wild resource amount rareness, are in endangered edge, and commodity cannot be provided maybe can only to provide a small amount of commodity.For wild licorice, the fifties in last century, the wild resource reserves of China were 2,000,000, and at present less than 350,000; The coverage of many local wild licorices drops to fragmentary distribution [10] from more than 90%.Therefore, the method for research saving Chinese medicaments material, while ensureing that medical industry and animal farming industry are to Chinese crude drug demand, the utilization rate improving Chinese crude drug is extremely urgent.
List of references
[1] Li Xiaobing, He little Juan, Liu Biao etc.; Angelica Polysaccharide is to the immunoregulation effect of H22 tumor-bearing mice; Journal of Chinese Integrative Medicine, 2010,8 (4): 363-367;
[2] Dan Tieying, Qiao Junhong, Yang Shuliang etc.; Lycium barbarum polysaccharide is on the impact of experimental esophageal carcinoma rat peripheral blood cell immunity immunologic function; Time precious traditional Chinese medical science traditional Chinese medicines, 2010,21 (4): 1016-1017;
[3] Qi Chunhui, Fu Yanrong, Zhang Yongxiang etc.; Liuwei Dihuang polysaccharides CA4-3 is to the effect of mouse B cell function. Chinese Pharmacological Bulletin, 2001,17 (4): 469-472.;
[4] Tingting ZHAO, Zhang Quanbin, Li Zhien etc.; Porphyra haitanensis polysaccharide and degradation products thereof are on the impact of immune cell propagation; New Chinese medicine and clinical pharmacology, 2009,20 (1): 30-32;
[5] Liu Yuan, Geng Yue; Ganoderan antineoplastic cell mechanism and the research of effect path; Food and pharmaceutical, 2008,10 (7): 48-51;
[6] Li Haibo, Mei Zhinan, Zhu Fan; The discussion of immune mechanism of anti-tumor by lycium barbarum polysaccharides; Journal of Chinese Hospital Pharmacy, 2005,25 (2): 115-7;
[7] Wang Xiangyan, Qi Yun, Cai Runlan etc.; The macrophage activation effect of Herba Cistanches polysaccharides; Chinese Pharmacological Bulletin, 2009,25 (6): 787-790;
[8]Paulsen B S.Plant polysaccharides with immunostimulatory activities.Curr Org Chem,2001,5(9):939-950；
[9] Wang Xiangyan, Qi Yun, Cai Runlan etc.; The macrophage activation effect of Herba Cistanches polysaccharides. Chinese Pharmacological Bulletin, 2009,25 (6): 787-790;
[10] prunus mume (sieb.) sieb.et zucc. gets the better of, Zhang Wensheng, Wang Yongyan; China's wild natural resources of Chinese medicinal materials protection is in urgent need of strengthening; China's Chinese medicine information magazine, 2008,15 (10): 4-6.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of vegetable polysaccharides oil emulsion and its production and use.
In order to solve the problems of the technologies described above, the invention provides a kind of preparation method of vegetable polysaccharides oil emulsion, Bao draws together as Xia Bu Sudden:
1), the preparation of oil phase:
Arlacel-80 and edible vegetable oil are mixed with volume ratio 10:90 ~ 16:84; Obtain oil phase;
That is, the volume content of Arlacel-80 in oil phase is 10 ~ 16% (being preferably 10 ~ 14%);
2), the preparation of aqueous phase:
Vegetable polysaccharides being dissolved in volumetric concentration is in the tween 80 aqueous solution of 5 ~ 7%, then regulates acid-base value to pH2 ~ 7 (being preferably pH6 ~ 7), obtains often milliliter of vegetable polysaccharides aqueous solution containing 8 ~ 64 milligrams of vegetable polysaccharidess, as aqueous phase;
3), by aqueous phase and oil phase fully mix with the volume ratio of 1:1 ~ 2, emulsifying, obtain vegetable polysaccharides oil emulsion.This vegetable polysaccharides oil emulsion is the oil emulsion that outward appearance is creamy white.
The present invention also provides simultaneously and utilizes said method to prepare and the vegetable polysaccharides oil emulsion that obtains.
The present invention also provides simultaneously and utilizes said method to prepare and a kind of purposes of vegetable polysaccharides oil emulsion of obtaining: as immunostimulant.
The present invention also provides simultaneously and utilizes said method to prepare and the another kind of purposes of vegetable polysaccharides oil emulsion that obtains: as the medicine of mammitis of cow.That is, the application of this vegetable polysaccharides oil emulsion in the medicine of preparation treatment mammitis of cow is provided.
Vegetable polysaccharides oil emulsion of the present invention is the oil emulsion that a kind of outward appearance containing vegetable polysaccharides, edible vegetable oil and surfactant is creamy white.
In the present invention:
Vegetable polysaccharides comprises astragalus polysaccharides, Rhizoma Atractylodis polysaccharide, Radix Codonopsis polysaccharide, Angelica Polysaccharide.
Vegetable oil comprises all edible plants oil, as Oleum Brassicae campestris, Semen Maydis oil, soybean oil, Oleum Camelliae, Oleum sesami, Oleum Arachidis hypogaeae semen, olive oil, Petiolus Trachycarpi oil, sunflower seed wet goods.
The adjustment of pH value belongs to routine techniques, and such as can adopt and drip concentration is that the hydrochloric acid of 1mol/L ~ 5mol/L or sodium hydroxide solution regulate.
Emulsifying refers to: mutually mixed by two kinds of mutual exclusive liquid, common method comprises strong stirring, or adds the surface tension that emulsifying agent changes one of them liquid, impels it can be uniformly distributed in another liquid.This also belongs to routine techniques.
The present invention has following technical advantage:
The invention provides a kind of preparation method of vegetable polysaccharides preparation, with only having the eighth vegetable polysaccharides of water preparation effective dose to make oil emulsion injection animal, and immune stimulation not being reduced.Said preparation can be used as the medicine of mammitis of cow, each case shot.Injection system and consumption are: at the oil emulsion of milch cow Ipsilateral lymphonodi mammarici position subcutaneous injection containing 8mg ~ 32mg vegetable polysaccharides; Or at the bilateral lymphonodi mammarici position subcutaneous injection of latent mammitis milch cow, every side injection is containing the oil emulsion of 16mg vegetable polysaccharides.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail.
Fig. 1 is that astragalus polysaccharides water preparation and oil emulsion subcutaneous injection affect comparison diagram to mouse spleen index;
Fig. 2 Radix Codonopsis polysaccharide, Angelica Polysaccharide, astragalus polysaccharides and Rhizoma Atractylodis polysaccharide oil emulsion and the rising action diagram of water preparation injection to Mouse interferon gamma;
Fig. 3 is astragalus polysaccharides water preparation and oil emulsion injection produce interferon gamma effect comparison diagram to mice.
Remarks illustrate: in figure, and the different numerical tabular of Superscript letters is shown with significant difference.
Detailed description of the invention
The preparation of embodiment 1, astragalus polysaccharides oil emulsion
1. the preparation of oil phase: by 1 milliliter of Arlacel-80 and 9 milliliters of edible rapeseed oil Homogeneous phase mixing, obtain oil phase;
2. the preparation of aqueous phase: be dissolved in by astragalus polysaccharides in the tween 80 aqueous solution of 6% (volume %), regulates acid-base value to pH7, makes every milliliter of aqueous solution containing 8 milligrams of astragalus polysaccharidess, as aqueous phase;
3. aqueous phase and oil phase are fully mixed according to volume ratio 1:1, emulsifying, namely, adopt high cut disperse emulsification homogenizer (b25 type, special electromechanical equipment Science and Technology Ltd. of Shanghai Bell product) stir 30 seconds under rotating speed 22000rpm, make that outward appearance is creamy white oil emulsion, obtain astragalus polysaccharides oil emulsion.
The preparation of embodiment 2, Angelica Polysaccharide oil emulsion
1. the preparation of oil phase: by 1 milliliter of Arlacel-80 and 9 milliliters of edible rapeseed oil Homogeneous phase mixing, obtain oil phase;
2. the preparation of aqueous phase: be dissolved in by Angelica Polysaccharide in the tween 80 aqueous solution of 5%, regulates acid-base value to pH6, makes every milliliter of aqueous solution containing 16 milligrams of Angelica Polysaccharide, as aqueous phase;
3. aqueous phase and oil phase are fully mixed according to volume ratio 1:1, emulsifying, make the oil emulsion that outward appearance is creamy white, obtain Angelica Polysaccharide oil emulsion.
Embodiment 3: the preparation of Radix Codonopsis polysaccharide oil emulsion
1. the preparation of oil phase: by 1 milliliter of Arlacel-80 and 9 milliliters of edible rapeseed oil Homogeneous phase mixing; Obtain oil phase;
2. the preparation of aqueous phase: be dissolved in by Radix Codonopsis polysaccharide in the tween 80 aqueous solution of 7%, regulates acid-base value to pH6, makes every milliliter of aqueous solution containing 32 milligrams of Radix Codonopsis polysaccharides, as aqueous phase;
3. aqueous phase and oil phase are fully mixed according to volume ratio 1:1, emulsifying, make the oil emulsion that outward appearance is creamy white, obtain Radix Codonopsis polysaccharide oil emulsion.
Embodiment 4: the preparation of Rhizoma Atractylodis polysaccharide oil emulsion
1. the preparation of oil phase: by 1 milliliter of Arlacel-80 and 9 milliliters of edible rapeseed oil Homogeneous phase mixing; Obtain oil phase;
2. the preparation of aqueous phase: be dissolved in by Rhizoma Atractylodis polysaccharide in the tween 80 aqueous solution of 5%, regulates acid-base value to pH6, makes every milliliter of aqueous solution containing 64 milligrams of Rhizoma Atractylodis polysaccharide, as aqueous phase;
3. aqueous phase and oil phase are fully mixed according to volume ratio 1:1, emulsifying, make the oil emulsion that outward appearance is creamy white, obtain Rhizoma Atractylodis polysaccharide oil emulsion.
Embodiment 5: various dose astragalus polysaccharides oil emulsion is to the immunostimulation of mice
Materials and methods
1. the preparation of astragalus polysaccharides oil emulsion
(1) preparation of oil phase: by 1 milliliter of Arlacel-80 and 9 milliliters of edible rapeseed oil Homogeneous phase mixing; Obtain oil phase;
(2) preparation of aqueous phase: be dissolved in by astragalus polysaccharides in the tween 80 aqueous solution of 6% (volume %), regulates acid-base value to pH 7, makes two kinds of aqueous solutions that every milliliter contains astragalus polysaccharides 5 milligrams and 10 milligrams, as aqueous phase;
(3) aqueous phase and oil phase are fully mixed according to volume ratio 1:1, emulsifying, make two kinds of astragalus polysaccharides oil emulsions that every milliliter contains astragalus polysaccharides 2.5 milligrams and 5 milligrams.
2. medication and sampling
By ICR male mice 40, be divided into 5 groups at random, often organize 8.5 groups of mices respectively subcutaneous shot 0.5ml normal saline, astragalus polysaccharides water preparation (containing astragalus polysaccharides 10mg), without medicine oil emulsion, astragalus polysaccharides oil emulsion 1 (containing astragalus polysaccharides 2.5mg), astragalus polysaccharides oil emulsion 2 (1.25mg).The 8th day after injection, blood sampling, prepared serum, detected serum interferon γ level; Put to death mice, weigh, get spleen and weigh, according to formulae discovery spleen index: heavy (the mg)/body weight (g) of spleen index=spleen.
Remarks illustrate:
The use cancelling astragalus polysaccharides in above-mentioned steps (2) is referred to without medicine oil emulsion; All the other are with the gains of above-mentioned preparation method.
Result and analysis
As shown in Figure 1, astragalus polysaccharides water preparation (10mg) and the spleen index without the subcutaneous injection mice generation of medicine oil emulsion are respectively 4.14 ± 0.61 and 5.51 ± 0.30; When astragalus polysaccharides and vegetable oil mixing, be prepared into astragalus polysaccharides oil emulsion 1 (containing astragalus polysaccharides 2.5mg) and astragalus polysaccharides oil emulsion 2 (1.25mg) injects mice afterwards, the spleen index of generation is respectively 6.45 ± 1.07 and 5.97 ± 0.68.Although the astragalus polysaccharides content in astragalus polysaccharides oil emulsion 1 (containing astragalus polysaccharides 2.5mg) is only 1/4th of water preparation (containing astragalus polysaccharides 10mg), the spleen index caused is significantly higher than astragalus polysaccharides or oil emulsion is used alone the spleen index (P<0.05) caused; Astragalus polysaccharides content in astragalus polysaccharides oil emulsion 2 (containing astragalus polysaccharides 1.25mg) is 1/8th of water preparation, but the spleen index caused is significantly higher than astragalus polysaccharides water preparation (P<0.05), and numerically higher than being used alone the spleen index (P>0.05) caused without medicine oil emulsion.The result shows, astragalus polysaccharides produces immunostimulation after being prepared into oil emulsion and is significantly higher than water preparation.
As shown in Figure 3, astragalus polysaccharides water preparation (10mg) and the serum interferon γ level produced without medicine oil emulsion subcutaneous injection mice and saline control group more do not raise; When astragalus polysaccharides and vegetable oil mixing, be prepared into astragalus polysaccharides oil emulsion 1 (containing astragalus polysaccharides 2.5mg) and astragalus polysaccharides oil emulsion 2 (1.25mg) injects mice afterwards, the serum interferon γ level of generation is significantly higher than astragalus polysaccharides water preparation and without medicine oil emulsion group (P<0.05).The result shows, the interferon gamma level that astragalus polysaccharides is induced after being prepared into oil emulsion is significantly higher than water preparation.
Embodiment 6: astragalus polysaccharides, Angelica Polysaccharide, Radix Codonopsis polysaccharide, Rhizoma Atractylodis polysaccharide Emulsion are on the impact of mice serum interferon
Materials and methods
56 ICR mices are divided into 8 groups at random, often organize 7.Every mice distinguishes water preparation or the oil emulsion of subcutaneous abdomen shot 0.2ml Rhizoma Atractylodis polysaccharide (1.6mg), astragalus polysaccharides (1.6mg), Angelica Polysaccharide (1.6mg) and Radix Codonopsis polysaccharide (1.6mg).Extract every Mouse Blood after 2 days in injection and be about 0.35ml, detect part serum interferon γ content by ELISA method.
Remarks illustrate:
One, the preparation method of Rhizoma Atractylodis polysaccharide oil emulsion is:
1. the preparation of oil phase: by 1 milliliter of Arlacel-80 and 9 milliliters of edible rapeseed oil Homogeneous phase mixing; Obtain oil phase;
2. the preparation of aqueous phase: be dissolved in by Rhizoma Atractylodis polysaccharide in the tween 80 aqueous solution of 5%, regulates acid-base value to pH7, makes every milliliter of aqueous solution containing 16 milligrams of Rhizoma Atractylodis polysaccharide, as aqueous phase;
3. aqueous phase and oil phase are fully mixed according to volume ratio 1:1, emulsifying, make the oil emulsion that outward appearance is creamy white, obtain Rhizoma Atractylodis polysaccharide oil emulsion.
Two, the preparation method of astragalus polysaccharides oil emulsion is:
1. the preparation of oil phase: by 1 milliliter of Arlacel-80 and 9 milliliters of edible rapeseed oil Homogeneous phase mixing; Obtain oil phase;
2. the preparation of aqueous phase: be dissolved in by astragalus polysaccharides in the tween 80 aqueous solution of 6% (volume %), regulates acid-base value to pH7, makes every milliliter of aqueous solution containing 16 milligrams of astragalus polysaccharidess, as aqueous phase;
3. aqueous phase and oil phase are fully mixed according to volume ratio 1:1, emulsifying, make the oil emulsion that outward appearance is creamy white, obtain astragalus polysaccharides oil emulsion.
Three, the preparation method of Angelica Polysaccharide oil emulsion is with embodiment 2.
Four, the preparation method of Radix Codonopsis polysaccharide oil emulsion is:
1. the preparation of oil phase: by 1 milliliter of Arlacel-80 and 9 milliliters of edible rapeseed oil Homogeneous phase mixing; Obtain oil phase;
2. the preparation of aqueous phase: be dissolved in by Radix Codonopsis polysaccharide in the tween 80 aqueous solution of 7%, regulates acid-base value to pH6, makes every milliliter of aqueous solution containing 16 milligrams of Radix Codonopsis polysaccharides, as aqueous phase;
3. aqueous phase and oil phase are fully mixed according to volume ratio 1:1, emulsifying, make the oil emulsion that outward appearance is creamy white, obtain Radix Codonopsis polysaccharide oil emulsion.
Result and being analyzed as follows:
As seen from Figure 2, the water preparation of mice shot Rhizoma Atractylodis polysaccharide, astragalus polysaccharides, Angelica Polysaccharide and Radix Codonopsis polysaccharide raises little to the interferon gamma level of mice serum, but the serum interferon γ level that the oil emulsion of injection four kinds of polysaccharide produces is significantly higher than water preparation, illustrate that polysaccharide oil emulsion produces immunostimulation and causes the effect of interferon gamma to be significantly higher than water preparation.
Embodiment 7: various dose Rhizoma Atractylodis polysaccharide oil emulsion Ipsilateral lymphonodi mammarici position subcutaneous injection is to the therapeutical effect of bovine subclinical mastitis
Object
Check various dose Rhizoma Atractylodis polysaccharide oil emulsion at Ipsilateral lymphonodi mammarici position subcutaneous injection to the therapeutical effect of bovine subclinical mastitis.
Materials and methods
1. Rhizoma Atractylodis polysaccharide oil emulsion: the preparation of (1) oil phase: by 1.4 milliliters of Arlacel-80s and 8.4 milliliters of edible rapeseed oil Homogeneous phase mixing; Obtain oil phase; (2) preparation of aqueous phase: be dissolved in by Rhizoma Atractylodis polysaccharide in the tween 80 aqueous solution of 6%, regulates acid-base value to pH 6, makes every milliliter of aqueous solution containing 24 milligrams of Rhizoma Atractylodis polysaccharide, as aqueous phase; (3) aqueous phase and oil phase are fully mixed according to volume ratio 1:2, emulsifying, make the oil emulsion that outward appearance is creamy white, obtain often milliliter of Rhizoma Atractylodis polysaccharide oil emulsion containing Rhizoma Atractylodis polysaccharide 8mg.
2. the sick cattle of latent mammitis is selected: to the newborn district filtering out the positive (++) and strong positive (+++) in the black-and-white flower cow in milk of cattle farm, the suburbs, Hangzhou with Hangzhou mastitis detectable (HMT), then the milk sample in aseptic collection breast district, low temperature is transported to laboratory and carries out milk somatic cell counting (SCC) and bacteria distribution qualification, interval one weekly check twice, filter out and have twice, Ge Ru district Somatic Cell Count SCC>50 ten thousand/ml at least, and the milch cow 24 of the bacteriology checking positive.
3, the treatment of latent mammitis milch cow: above 24 cow heads are divided into 4 groups at random, often organizes 6.Group 1 ~ 3 is respectively at ill Ru Qu side lymphonodi mammarici subcutaneous shot Rhizoma Atractylodis polysaccharide oil emulsion 1ml (8mg), 2ml (16mg) or 4ml (32mg); Matched group is not treated by milch cow.
4. treat post-sampling: after medication 1,2,3 week, aseptic collection is tested newborn district milk sample and is carried out Somatic Cell Count, milk sample bacteriology checking and NAGase determination of activity.
5. latent mammitis detects (HMT method): with hot towel scrubber wash clean each newborn district by cattle to be checked, discard three milk, respectively the breast in 4 Ge Ru districts is squeezed 4 little indoor at diagnosis dish, each cell is about 2ml containing milk, add 2ml HMT diagnostic reagent respectively, diagnosis dish is shaken, result of determination (criterion is in table 1) in concentric circular.
The criterion of table 1 HMT diagnostic reagent
6. milk somatic cell (SCC) counting: carry out according to instrument description.First with standard substance, instrument is corrected, then by milk sample heating in water bath to 40-42 DEG C, detect after shaking up, record data.
7. milk bacteriology checking: carry out according to NMC (National Mastitis Council) recommended program.Concrete operation step is as follows:
(1) get 10 μ l milk samples and be inoculated in 6% Sheep Blood plate, put 37 DEG C of incubators and cultivate 24 ~ 48 hours.
(2) observe colony morphology characteristic, picking colonies typical is inoculated in ordinary broth and carries out Zengjing Granule, 37 DEG C of shaken cultivation 24 hours.Smear, Gram’s staining, microscopy, carry out preliminary judgement.
(3) biochemical identification is carried out.First catalase test is carried out to gram-positive cocci and determine that this bacterium is staphylococcus or streptococcus.With coagulase test of blood plasma, staphylococcus is divided into staphylococcus aureus and coagulase negative staphylococcus (CNS); With tests such as CAMP test, Esculin hydrolysis, sodium hippurate hydrolysis, the reduction of methylene blue Lac Bovis seu Bubali, streptococcus is divided into streptococcus agalactiae, streptococcus dysgalactiae or streptococcus uberis.What do not belong to above-mentioned five kinds of antibacterials is classified as other class.
8. milk NAGase Enzyme assay: operate according to the following steps:
(1) by putting in refrigerator multigelation containing the centrifuge tube of 5ml milk 3 times, the NAGase in born of the same parents is fully discharged.
(2) by the milk sample of having melted at the centrifugal 20min of 4000rpm, removing upper strata butterfat.Then in the milk of defat, drip 10% glacial acetic acid, regulate pH to 4.6, leave standstill in room temperature; Then 4000rpm, centrifugal 20min, separate out milk surum.
(3) the NAGase activity in milk surum is detected according to test kit description.Concrete operations are: in blank tube, add distilled water (0.1ml); Paranitrophenol titer (0.6mmol/L, 0.1ml) is added at standard pipe; 0.1ml milk surum to be measured is respectively added in control tube and mensuration pipe.0.5ml reagent one is respectively added in blank tube and standard pipe; 0.5ml substrate buffer solution is added in mensuration pipe.Each pipe adds 2ml reagent three after putting 37 DEG C of water-bath 15min; 0.5ml substrate buffer solution is added in control tube.Finally in each test tube, respectively add 0.05ml reagent four, mixing, measure OD value at 400nm place.Active according to formulae discovery NAGase: diluted sample multiple before NAGase vigor (U/L)=(measuring pipe OD value-control tube OD value)/(standard pipe OD value-blank tube OD value) × standard concentration (0.6mmol/L) × 1/15 × 1000 × test in milk surum
9. statistical analysis: data Mean ± S.E. represents.Check the diversity compared between the group same period, before and after medication in group with t, before statistical analysis, SCC, NAGase numerical value is carried out logarithmic transformed.
Results and analysis
1. the treatment of Rhizoma Atractylodis polysaccharide oil emulsion reduces milk somatic cell content
Milk somatic cell number before treatment between each group is without significant difference, after the treatment of Rhizoma Atractylodis polysaccharide oil emulsion, somatic number in three treatment group milk declines all to some extent, wherein the milk somatic cell number for the treatment of group 2 (Rhizoma Atractylodis polysaccharide breast 16mg) after the treatment 2,3 weeks significantly lower than concurrent control group, also have significant difference compared with before treating; And matched group milk somatic cell number before the treatment after substantially constant (table 2).
Table 2 is each group milk sample somatic number (average × 10 before and after treating
4± S.E./ml)
before representing treatment; A represents that treatment group is compared with concurrent control group significant difference (P<0.05); * significant difference (P<0.05) and extremely remarkable (P<0.01) compared with before treatment is represented respectively with * *.
2. the treatment of Rhizoma Atractylodis polysaccharide oil emulsion reduces the activity of milk NAGase enzyme
Before treatment, the NAGase enzymatic activity of each group milk is without significant difference, after the treatment of Rhizoma Atractylodis polysaccharide oil emulsion, NAGase activity in three treatment group milk declines all to some extent, wherein treatment group 2 (16mg) milk NAGase activity after the treatment 2,3 weeks significantly lower than before treatment, after the treatment 1,2,3 week remarkable in matched group; Treatment group 3 (32mg) milk NAGase activity after the treatment 2 weeks significantly lower than matched group.
The NAGase activity (average ± S.E.U/L) of table 3. each group milk sample before and after treating
before representing treatment; A represents that treatment group compares significant difference (P<0.05) (P<0.01) with concurrent control group; * significant difference (P<0.05) and extremely remarkable (P<0.01) compared with before treatment is represented respectively with * *
This result of study shows a subcutaneous injection Rhizoma Atractylodis polysaccharide oil emulsion at the lymphonodi mammarici position of latent mammitis milch cow Ipsilateral, significantly can reduce the activity of Somatic Cells Content in latent mammitis breast district milk and NAGase enzyme.Somatic Cells Content during milch cow trouble mastitis in milk and the activity of NAGase enzyme raise.Injection Rhizoma Atractylodis polysaccharide Emulsion can make the Somatic Cells Content in Bovine Mastitis Milk district milk and NAGase activity reduce, and illustrates that Rhizoma Atractylodis polysaccharide oil emulsion has therapeutical effect to bovine subclinical mastitis.And inject containing 64mg Rhizoma Atractylodis polysaccharide water preparation in contrast time (experiment method is the same), the therapeutic effect of this matched group to bovine subclinical mastitis is nothing like the therapeutic effect of " treatment group 1 (8mg) " of the present invention.
Embodiment 8: Rhizoma Atractylodis polysaccharide oil emulsion bilateral lymphonodi mammarici position subcutaneous injection is to the therapeutical effect of bovine subclinical mastitis
Materials and methods
1. Rhizoma Atractylodis polysaccharide oil emulsion preparation, the sick cattle of latent mammitis are selected, latent mammitis detects (HMT method), milk somatic cell (SCC) counting, milk bacteriology checking, milk NAGase Enzyme assay with embodiment 7.
2. the treatment of latent mammitis milch cow: the latent mammitis milch cow picked out is divided into two groups at random: treatment group, 11 cow heads (having 14 to suffer from latent mammitis in 41 Ge Ru districts), at the lymphonodi mammarici position subcutaneous shot Rhizoma Atractylodis polysaccharide oil emulsion of milch cow, every side 2ml (16mg); Matched group, 11 cow heads (having 12 to suffer from latent mammitis in 44 Ge Ru districts), do not treat.
3. treat post-sampling: after medication 1,2,3 week, aseptic collection is tested newborn district milk sample and is carried out Somatic Cell Count, milk sample bacteriology checking and NAGase determination of activity.
4. statistical analysis: data Mean ± S.E. represents.Check the diversity compared between the group same period, before and after medication in group with t, before statistical analysis, SCC, NAGase numerical value is carried out logarithmic transformed.
Results and analysis
1, Rhizoma Atractylodis polysaccharide oil emulsion bilateral lymphonodi mammarici position subcutaneous injection reduces the Somatic Cells Content of ill newborn district milk:
The milk somatic cell number for the treatment of first two groups without significant difference, after the treatment of Rhizoma Atractylodis polysaccharide oil emulsion, treatment group milk somatic cell number after the treatment 2,3 weeks significantly lower than concurrent control group, also significantly reduce (table 4) compared with before treating.
Change (average × 10 of ill newborn district milk sample somatic number before and after the treatment of table 4 Rhizoma Atractylodis polysaccharide oil emulsion
4± S.E./ml)
treat the newborn district that front twice inspection milk somatic cell number is all greater than 500,000/ml and the bacteriology checking positive.
#before treatment.
ap<0.05, and concurrent control group compares.
* compare before P<0.05 and group internal therapy.
2, Rhizoma Atractylodis polysaccharide oil emulsion bilateral lymphonodi mammarici position subcutaneous injection reduces the NAGase enzymatic activity of ill newborn district milk:
The NAGase enzymatic activity for the treatment of front two groups of milk without significant difference, after the treatment of Rhizoma Atractylodis polysaccharide oil emulsion, treatment group milk NAGase activity after the treatment 2,3 weeks significantly lower than concurrent control group, after the treatment 1,2,3 week remarkable in before treating.
Change (average × 10 of ill newborn district milk sample NAGase enzymatic activity before and after the treatment of table 5 Rhizoma Atractylodis polysaccharide oil emulsion
4± S.E./ml)
treat the newborn district that front twice inspection milk somatic cell number is all greater than 500,000/ml and the bacteriology checking positive.
#before treatment.
ap<0.05, and concurrent control group compares.
* P<0.05, P<0.01, and compare before group internal therapy.
3. Rhizoma Atractylodis polysaccharide oil emulsion bilateral lymphonodi mammarici position subcutaneous injection reduces the somatic number of newborn district mixing milk
The somatic number for the treatment of front two groups of milch cow four newborn district mixing milks is without significant difference, and after the treatment of Rhizoma Atractylodis polysaccharide oil emulsion, the SCC for the treatment of group milch cow is significantly lower than the somatic number (table 6) before concurrent control group and group internal therapy.
Table 6 Rhizoma Atractylodis polysaccharide oil emulsion treats the somatic number (average ± S.E.10 of front rear milk area mixing milk
4/ ml)
| Group | Milch cow number | Before treatment | After treatment |
| Rhizoma Atractylodis polysaccharide oil emulsion | 11 | 62.55±25.38 | 29.21±6.85 a* |
| Matched group | 11 | 56.66±32.34 | 54.89±11.88 |
ap<0.05, and concurrent control group compares.
* P<0.05, and compare before group internal therapy.
4. Rhizoma Atractylodis polysaccharide oil emulsion bilateral lymphonodi mammarici position subcutaneous injection reduces the quantity in bacteriological infection breast district
Treatment group You21Ge Ru district bacteriological infection before treatment, matched group You23Ge Ru district bacteriological infection, without significant difference between two groups.After the treatment of Rhizoma Atractylodis polysaccharide oil emulsion, the infection breast district number for the treatment of group is reduced to 9 (P<0.05), and the infection of concurrent control group breast district is still more than 20, does not reduce (table 7).
Bacterioscopy positive udder region number (tested newborn district number: treatment group=41 before and after the treatment of table 7, Rhizoma Atractylodis polysaccharide oil emulsion; Matched group=44)
#before treatment.
* P<0.05, and compare before group internal therapy.
This result of study show latent mammitis milch cow at bilateral lymphonodi mammarici position a subcutaneous subcutaneous injection Rhizoma Atractylodis polysaccharide oil emulsion, significantly can reduce the Somatic Cells Content of milk, the activity of NAGase enzyme and the bacterial infections levels of mammary gland, illustrate that Rhizoma Atractylodis polysaccharide oil emulsion has therapeutical effect to bovine subclinical mastitis.
Finally, it is also to be noted that what enumerate above is only several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be had.All distortion that those of ordinary skill in the art can directly derive from content disclosed by the invention or associate, all should think protection scope of the present invention.
Claims (5)
1. the preparation method of vegetable polysaccharides oil emulsion, is characterized in that Bao draws together as Xia Bu Sudden:
1), the preparation of oil phase:
Arlacel-80 and edible vegetable oil are mixed with volume ratio 10:90 ~ 16:84; Obtain oil phase;
2), the preparation of aqueous phase:
Vegetable polysaccharides being dissolved in volumetric concentration is in the tween 80 aqueous solution of 5 ~ 7%, then regulates acid-base value to pH2 ~ 7, obtains often milliliter of vegetable polysaccharides aqueous solution containing 8 ~ 64 milligrams of vegetable polysaccharidess, as aqueous phase;
3), by aqueous phase and oil phase fully mix with the volume ratio of 1:1 ~ 2, emulsifying, obtain vegetable polysaccharides oil emulsion.
2. the preparation method of vegetable polysaccharides oil emulsion according to claim 1, is characterized in that:
Described vegetable polysaccharides is astragalus polysaccharides, Rhizoma Atractylodis polysaccharide, Radix Codonopsis polysaccharide or Angelica Polysaccharide.
3. method preparation and the vegetable polysaccharides oil emulsion that obtains as claimed in claim 1 or 2.
4. method preparation and the purposes of vegetable polysaccharides oil emulsion that obtains as claimed in claim 1 or 2, is characterized in that: as immunostimulant.
5. method preparation and the purposes of vegetable polysaccharides oil emulsion that obtains as claimed in claim 1 or 2, is characterized in that: as the medicine of mammitis of cow.
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