CN103142742B - Method for preparing oral solution of poplar flower - Google Patents
Method for preparing oral solution of poplar flower Download PDFInfo
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Abstract
The invention discloses a method for preparing an oral solution of the poplar flower and belongs to the technical field of veterinary medicaments. The method comprises the following steps of: (1) putting the poplar flower into an extraction tank, adding water into the poplar flower, stirring, and increasing the temperature to 45-50 DEG C, wherein the water is 4-6 times of the mass of the poplar flower; (2) adding cellulose into the extraction tank, adjusting the pH value of the materials to 5.0-5.5, and hydrolyzing under the action of the cellulose for 90-120 minutes at the temperature of 45-50 DEG C, wherein the mass of the cellulose is 3-7% of that of the poplar flower; and (3) filtering a solution of enzymolysis, and collecting a filtrate to obtain the poplar-flower oral solution. The poplar flower is extracted by adopting the technology of hydrolyzing under the action of the cellulose, and a novel process for extracting the poplar flower is developed, thus the time spent in extracting the poplar flower is shortened, the heat consumption is reduced, the amount of water used in the process is reduced, and the purposes of saving energy, reducing emission, improving the work efficiency and the extraction efficiency are achieved. The method has good social and economic benefits.
Description
Technical field
The present invention relates to veterinary drug technical field, especially a kind of preparation method of poplar flower oral liquid.
Background technology
Flower of poplar is Salicaceous Plants Canada willow populus canadensis Mench. inflorescence, is rich in the compositions such as flavone, alkaloid, organic acid, vitamin, phenols, has the effect of strengthening the spleen and nourishing the stomach, removing dampness dysentery relieving, relieving diarrhea with astringents, heat-clearing and toxic substances removing.Poplar flower oral liquid is recorded in bis-of 2010 editions < < Chinese veterinary pharmacopoeia > >, clinical pig, sheep, the fowl dysentery of being used for.Modern pharmacology research shows that flower of poplar has the effects such as antibacterial, antipyretic and anti-inflammatory, dysentery relieving antidiarrheal.Tradition poplar flower oral liquid is to be prepared from by immersion, heated and boiled, decoction, the method such as concentrated, and the process time is long, and thermal energy consumption is high, process water consumption is large, extraction ratio is low.1 ton of oral liquid of every production needs more than tens hour from soaking, decoct, concentrate, precipitate, be filled into subpackage; Decoction, the concentrated hectic fever steam 3-5 ton that needs; Medical material decocts 2 times, need to consume 8 tons of purified water (needing 40 tons by drinking water standard), concentrated 10 tons of the waters that need; Effective ingredient flavones content is up to 0.132%.
Middle the effective elements of the medicine stores in the framework that is cell wall cellulose formation mostly, in being wrapped in, extracts its effective ingredient by cellulose, and the cellulose framework that must first break its cell wall is fully dissolved out effective ingredient.For Chinese medicine extraction research more be cellulase, cellulose is to be connected with Isosorbide-5-Nitrae-β glucoside bond by β-D-Glucose, with cellulase degradation, can destroy β-D-Glucose key, makes plant cell wall destruction, is conducive to the extraction to effective ingredient.In recent years, the report of domestic employing cellulase extraction Effective Component of Chinese Medicine constantly occurs.The introduction employing enzyme hydrolysis methods such as Lv Weiming extract separated baicalin and reach 75.67% from Radix Scutellariae, and yield is 2.46%.Someone extracts total flavones under the effect of cellulase from Radix Puerariae, and yield has improved 13%.In Radix Notoginseng leaching process, with cellulase degradation effect, destroy cell wall, the total soap of Radix Notoginseng former times extraction ratio improves 23.5%.In addition, first Chinese medicine one factory in Shanghai applies enzyme process and has successfully prepared SHENGMAI YIN KOUFUYE.Have and improve yield, energy savings with traditional extraction process comparison cellulase solution, reduce solvent use amount etc. advantage, but so far there are no, adopt the report of cellulase extraction flower of poplar.
Summary of the invention
The invention provides a kind of preparation method of poplar flower oral liquid, utilize cellulase hydrolysis technology to extract flower of poplar, developed a kind of new technology of extracting flower of poplar, greatly shorten extraction time, reduced thermal energy consumption, reduced process water consumption, reach energy-saving and emission-reduction, increased work efficiency and the object of extraction ratio, there is good society and economic benefit.
A preparation method for poplar flower oral liquid, comprises the steps:
(1) flower of poplar is put into extraction pot, add flower of poplar quality 4-6 water doubly, stir and be warming up to 45-50 ℃;
(2) in extraction pot, add the cellulase of flower of poplar quality 3-7%, regulate pH value to 5.0-5.5, enzymolysis 90-120min at 45-50 ℃;
(3) enzymolysis solution is filtered, collect filtrate, obtain poplar flower oral liquid.
In step (2), with the lactic acid solution of 10-50% or the sodium hydroxide solution of 10-50%, regulate pH value.
The quality of step (2) cellulase is 4% of flower of poplar quality.
In step (2), enzymolysis time is 120min.
Its usage and dosage is: horse, cattle 50～100ml; Sheep, pig 10～20ml; Rabbit, fowl 1～2ml.
Single factors such as enzyme addition, hydrolysis temperature, enzymolysis time, solvent pH value affect test to flavone extraction ratio:
1, cellulase activity detects
Cellulase is a kind of important compound bio-enzyme, mainly circumscribed 1,4 beta-glucanase, Endo-β-glucanase and beta-glucosidase etc., consists of, and also has the xylanase of very high vigor.
Cellulose and hemicellulose have formed plant cell wall framework, and flower of poplar effective ingredient is present in the framework that has cellulose and hemicellulose formation.Cellulase has specificity, cellulase decomposition of cellulose, and xylanase decomposes hemicellulose, and glucanase continues to resolve into glucose by cellulose and hemicellulose catabolite.Flower of poplar cell wall under cellulase and xylanase effect fully decomposes and discharges flower of poplar effective ingredient.
Adopt spectrophotometry cellulase and Xylanase activity.
Detection fibers element enzyme cellulase >=30000U/g, xylanase >=100000U/g, 1,4 beta-glucanase >=120000U/g.
2, standard solution preparation and Specification Curve of Increasing
The preparation of 2.1 control substance of Rutin solution:
Data demonstration flavone detects take rutin as standard reference material.
Accurately take control substance of Rutin 10mg, be placed in 50ml volumetric flask, add 30% ethanol 30ml, ultrasonic concussion makes it to dissolve, and is cooled to room temperature, and is diluted to scale, shakes up to such an extent that concentration is the control substance of Rutin solution of 200 μ g/ml, and cold preservation is standby.
The selection of 2.2 absorbing wavelength:
Reference substance is measured determining of wavelength: get reference substance solution 1ml, by the method under standard curve item, undertaken, in the interscan of 400～700nm wave-length coverage, result is absorption maximum at 500nm wavelength place.Determining of sample determination wavelength: the ethanol extract 1ml of sample thief, by the method under standard curve item, undertaken, in the interscan of 400～700nm wave-length coverage, result is absorption maximum at 500nm wavelength place.
2.3 Specification Curve of Increasings:
Precision takes control substance of Rutin solution 0,1.0,2.0,3.0,4.0,5.0ml is respectively to 25ml volumetric flask, add respectively 5% sodium nitrite solution 0.3ml, shake up, place 6min, add 10% aluminum nitrate solution 0.3ml, place 6min, add 1mol sodium hydroxide solution 4ml, use respectively 30% ethanol standardize solution, shake up, place 15min, put in cuvette, at 500nm wavelength place, measure absorbance.Take absorbance A as vertical coordinate, and concentration C is abscissa, drawing standard curve (see figure 1).Through returning statistics, obtain standard curve equation: A=0.0486+0.0002 R
2=0.9996.Concentration within the scope of 0-400 μ g/ml R square reach 0.9996, linear relationship is good.
3, single-factor influence test
3.1 cellulase additions are on the impact of flavone extraction ratio in poplar flower oral liquid (A factor)
Get 7 parts of flower of poplar, every part of 100g, cellulase addition is respectively 2g, 3g, 4g, 5g, 6g, 7 g, 8 g, hydrolysis temperature is controlled at 45 ℃, enzymolysis pH value and is controlled under 5.0 conditions, enzymolysis time is controlled at 90 min, collect enzymolysis solution, according to the assay method that detects general flavone content in poplar flower oral liquid, detect general flavone content, cellulase addition is shown in Fig. 2 to the impact of flavone extraction ratio in poplar flower oral liquid.
As shown in Figure 2, enzyme addition single-factor influence result of the test shows, get 100g flower of poplar and prepare poplar flower oral liquid, along with enzyme addition increases, in oral liquid, flavone extraction ratio raises, flavone extraction ratio in sample when enzyme adds 3g to, than the extraction ratio that adds 2g, significantly raise, while adding 4-8g to, flavone extraction ratio improves always, adding the 7g amplitude that flavone extraction ratio improves when above to diminishes, the many factors such as flavone extraction ratio in comprehensive production cost and flower of poplar, get 100g flower of poplar and prepare oral liquid, add 3-7g cellulase more suitable, be preferably 4g.
3.2 hydrolysis temperatures are on the impact of flavone extraction ratio in poplar flower oral liquid (B factor)
Get 5 parts of flower of poplar, every part of 100g, cellulase addition is 4g, hydrolysis temperature is respectively 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, enzymolysis pH value is controlled under the conditions such as 5.0, and enzymolysis time is controlled at 90 min, collects enzymolysis solution, according to the assay method that detects general flavone content in poplar flower oral liquid, detect general flavone content, hydrolysis temperature is shown in Fig. 3 to the impact of flavone extraction ratio in poplar flower oral liquid.
As shown in Figure 3, under same enzyme addition, enzymolysis time and enzymolysis pH condition, hydrolysis temperature single-factor influence result of the test shows, hydrolysis temperature is from 35 ℃ to 45 ℃, with flavone extraction ratio in temperature rising poplar flower oral liquid, by 0.102%, be elevated to 0.154%, the hydrolysis temperature extraction ratio 0.144% 50 ℃ time that raises, when hydrolysis temperature reaches 55 ℃, extraction ratio drops to 0.016%, in view of this, hydrolysis temperature impact in cellulose enzyme process extracts is larger, and the temperature of suitable extraction is between 45 ℃ to 50 ℃.
Enzymolysis time is on the impact of flavone extraction ratio in poplar flower oral liquid (C factor)
Get flower of poplar 500g, be divided into 5 parts, every part of 100g, cellulase addition is 4g, 45 ℃ of hydrolysis temperatures, enzymolysis time is respectively 30min, 60 min, 90 min, 120 min, 150 min, enzymolysis pH value is controlled at 5.0, and enzymolysis time arrives, and collects enzymolysis solution, according to the assay method that detects general flavone content in poplar flower oral liquid, detect general flavone content, enzymolysis time is shown in Fig. 4 to the impact of flavone extraction ratio in poplar flower oral liquid.
As shown in Figure 4, under same enzyme addition, hydrolysis temperature and enzymolysis pH condition, enzymolysis time single-factor influence result of the test shows: from the enzymolysis 30min time, start to sample, with enzymolysis time, extending extraction ratio improves constantly, after enzymolysis time reaches 120min, extraction ratio is substantially constant, and the extraction ratio of enzymolysis 150min and 120min is basically identical.Therefore, from single factor experiment, enzymolysis time is more suitable at 90-120min, is preferably 120min.
3.4 enzymolysis pH value are on the impact of flavone extraction ratio in poplar flower oral liquid (D factor)
Get flower of poplar 500g, be divided into 5 parts, every part of 100g, cellulase addition is 4g, 45 ℃ of enzymolysis time 90 min of hydrolysis temperature, pH value is respectively 4.0,4.5,5.0,5.5,6.0, collects enzymolysis solution, according to the assay method that detects general flavone content in poplar flower oral liquid, detect general flavone content, enzymolysis pH value is shown in Fig. 5 to the impact of flavone extraction ratio in poplar flower oral liquid.
As shown in Figure 5, under the condition of same enzyme addition, hydrolysis temperature and enzymolysis time, enzymolysis pH value single-factor influence result of the test shows: enzymolysis pH value flavone extraction ratio in 5.0,5.5 o'clock poplar flower oral liquids is the extraction ratio of 4.0,4.5,6.0 o'clock apparently higher than enzymolysis pH value, and wherein enzymolysis pH value is that to be starkly lower than pH value be 4.0 extraction ratio 0.082% for the extraction ratio 0.023% of 6.0 o'clock.In view of this pH value is that 6.0 pairs of cellulase destroy seriously, causes cellulase inactivation, has affected hydrolysis result.Therefore, in cellulase solution extraction poplar flower oral liquid technique, setting enzymolysis pH scope is 5.0-5.5.
4, orthogonal experiments:
Select L9(3
4) orthogonal array take flavone extraction ratio as performance assessment criteria, investigate cellulase solution and extract enzyme addition factor A, hydrolysis temperature factor B, enzymolysis time factor C, the impact of enzymolysis pH value factor D on poplar flower oral liquid extraction process in poplar flower oral liquid, orthogonal experiments is in Table 1.
Table 1 L9(3
4) orthogonal array
By to L9(3
4) orthogonal experiments carries out range analysis, the impact of flavone extraction ratio is sequentially followed successively by A>B>C>D affecting the A factor enzyme addition of flavone extraction ratio, B factor hydrolysis temperature, C factor enzymolysis time and D factor enzymolysis pH value.In A factor, kA3 is almost consistent with kA2, and kA3, kA2 and KA1 difference are obvious aobvious; In B factor, KB2 and KB3 difference are little, both and KB1 obvious difference; In C factor, kC3 is apparently higher than kC1 and kC2; KD3 and kD2 are more approaching, apparently higher than KD1.
Comprehensive above factor influence degree and controlling factors ability, addition and the enzymolysis time of enzyme are easily controlled, and are not subject to other condition impacts, and hydrolysis temperature and enzymolysis pH value are distributed with different in extracting solvent.Therefore, by quadrature L9(3
4) filter out cellulase solution extraction flower of poplar process conditions for adding enzyme 3-7g, enzymolysis time 90-120min, hydrolysis temperature is controlled between 45-50 ℃, and enzymolysis pH value is controlled between 5.0-5.5.
5, demonstration test
According to L9(34) the interpolation enzyme 3-7% that filters out of orthogonal test, enzymolysis time 90-120min, hydrolysis temperature is controlled between 45-50 ℃, enzymolysis pH value is controlled at the process conditions between 5.0-5.5, at pilot plant, carried out the proving test of continuous three batches, three batches of test temperatures are respectively 46 ℃, 48 ℃, 48 ℃, and pH is respectively 5.2,5.5,5.1, three batches of test products flavone extraction ratios are respectively 0.185%, 0.189%, 0.188%, and weighted average demonstration test extraction ratio is 0.187%.According to bis-poplar flower oral liquid quality standard checks of 2010 editions < < Chinese veterinary pharmacopoeia > >, every assay is up to specification.
6, clinical efficacy:
Drug sensitive test result shows that cellulase solution extraction poplar flower oral liquid of the present invention has obvious fungistatic effect to broiler escherichia coli, can be used for the treatment of broiler colibacillosis.
6.1 poplar flower oral liquids are to the colibacillary drug sensitive test of broiler.Seminar adopts the pathological material of disease be positioned at the colibacillosis that the chicken house clinical onset of three regions makes a definite diagnosis to be numbered A, B, C, the poplar flower oral liquid of the present invention of take is numbered 1 as trial drug susceptibility sheet, the former powder of the florfenicol of take, the former powder of colistin sulfate, the former powder of polygynax, the former powder in amoxicillin, as control drug (being numbered 2,3,4,5), have been carried out drug sensitivity test.Result of the test is in Table 2:
Table 2 drug sensitivity test result (unit: mm)
Result of the test by table 2 shows: from three groups of chicken groups' of different geographical bacterial strain, no matter whether medication was treated, all responsive to poplar flower oral liquid.
The clinical effect trial of 6.2 poplar flower oral liquid control broiler colibacillosis
12600 plumages that project team raises for certain broiler breeding field, 15 ages in days, the colibacillosis of the clinical generation of Ross 308 Broiler has been carried out clinical efficacy contrast test.This test is control drug with poplar flower oral liquid and 10% colistin sulfate soluble powder and 10% ofloxacin soluble powder, investigates cellulase hydrolysis poplar flower oral liquid clinical efficacy, and result of the test is in Table 3.
Table 3 poplar flower oral liquid clinical efficacy
Result of the test by table 3 shows: it is extremely remarkable compared with C group, D group difference that A group, B organize death rate, and there were significant differences in weightening finish, and feedstuff-meat ratio is without significant difference; Dead number, death rate, weightening finish, the material meat test effect washed in a pan of A group is significantly better than B group, and single statistical data is without significant difference.
7, Economic and Efficiency Analysis
Through experimental analysis, adopt enzymolysis process to prepare poplar flower oral liquid, steam consumption declines 86.4%, and using water wisely 53.5%, has saved ample resources.
45 tons of every production poplar flower oral liquids, are 17.5 ten thousand yuan by price per ton, 787.5 ten thousand yuan of new output values, and after 97.058 ten thousand yuan of the newly-increased manufacturing cost of deduction and development expenses, newly-increased net earnings is 690.442 ten thousand yuan; Its input-output ratio is 1:8.11, and scientific and technological investment yield is that 1:7.11 popularization investment yield is 1:40.28.In this project, 10000 yuan of the every increases of social investment, can increase by 81100 yuan of outputs, can return 71100 yuan of net earnings; Promote 10000 yuan of the every increases of specific investment cost, can make society obtain 402800 yuan of net earnings.Therefore, implement this project and obtained very significant society and economic benefit.
The beneficial effect that adopts technique scheme to produce is:
1. the present invention utilizes cellulase hydrolysis technology to extract flower of poplar, developed a kind of new technology of extracting flower of poplar, shorten the process time, reduced thermal energy consumption, reduced process water consumption, reach energy-saving and emission-reduction, increased work efficiency and the object of extraction ratio, there is good society and economic benefit.
2. the extraction ratio of flavone is brought up to more than 0.18%, than traditional handicraft, improved more than 40%.
Accompanying drawing explanation
Below in conjunction with the drawings and specific embodiments, the present invention is further detailed explanation.
Fig. 1 is the canonical plotting of control substance of Rutin;
Fig. 2 is that the addition of enzyme affects schematic diagram to flavone extraction ratio;
Fig. 3 is that hydrolysis temperature affects schematic diagram to flavone extraction ratio;
Fig. 4 is that enzymolysis time affects schematic diagram to flavone extraction ratio;
Fig. 5 is that enzymolysis pH value affects schematic diagram to flavone extraction ratio.
The specific embodiment
The cellulase using in following examples is provided by Kang Yuan bio tech ltd, Shandong.
Embodiment 1
(1) 100g flower of poplar is put into extraction pot, add flower of poplar quality 400g water, stir and be warming up to 45 ℃;
(2) 5g cellulase in extraction pot, regulates pH value to 5.5, enzymolysis 95min at 45 ℃;
(3) enzymolysis solution is filtered, collect filtrate, obtain poplar flower oral liquid 105mL.
According to the assay method that detects general flavone content in poplar flower oral liquid, detect general flavone content in the present embodiment, the extraction ratio of total flavones is as calculated: 0.188%.According to bis-poplar flower oral liquid quality standard checks of 2010 editions < < Chinese veterinary pharmacopoeia > >, every assay is up to specification.
(1) 100g flower of poplar is put into extraction pot, add flower of poplar quality 500g water, stir and be warming up to 45-50 ℃;
(2) in extraction pot, add 3g cellulase, regulate pH value to 5.1, enzymolysis 110min at 46 ℃;
(3) enzymolysis solution is filtered, collect filtrate, obtain poplar flower oral liquid 120mL.
According to the assay method that detects general flavone content in poplar flower oral liquid, detect general flavone content in the present embodiment, the extraction ratio of total flavones is as calculated: 0.181%.According to bis-poplar flower oral liquid quality standard checks of 2010 editions < < Chinese veterinary pharmacopoeia > >, every assay is up to specification.
(1) 100g flower of poplar is put into extraction pot, add flower of poplar quality 600g water, stir and be warming up to 45-50 ℃;
(2) in extraction pot, add 7g cellulase, regulate pH value to 5.4, enzymolysis 100min at 48 ℃;
(3) enzymolysis solution is filtered, collect filtrate, obtain poplar flower oral liquid 130mL.
According to the assay method that detects general flavone content in poplar flower oral liquid, detect general flavone content in the present embodiment, the extraction ratio of total flavones is as calculated: 0.189%.According to bis-poplar flower oral liquid quality standard checks of 2010 editions < < Chinese veterinary pharmacopoeia > >, every assay is up to specification.
Embodiment 4
(1) 100g flower of poplar is put into extraction pot, add flower of poplar quality 450g water, stir and be warming up to 45-50 ℃;
(2) in extraction pot, add 4g cellulase, regulate pH value to 5.2, enzymolysis 120min at 50 ℃;
(3) enzymolysis solution is filtered, collect filtrate, obtain poplar flower oral liquid 108mL.
According to the assay method that detects general flavone content in poplar flower oral liquid, detect general flavone content in the present embodiment, the extraction ratio of total flavones is as calculated: 0.186%.According to bis-poplar flower oral liquid quality standard checks of 2010 editions < < Chinese veterinary pharmacopoeia > >, every assay is up to specification.
(1) 100g flower of poplar is put into extraction pot, add flower of poplar quality 500g water, stir and be warming up to 45-50 ℃;
(2) in extraction pot, add 6g cellulase, regulate pH value to 5.0, enzymolysis 90min at 46 ℃;
(3) enzymolysis solution is filtered, collect filtrate, obtain poplar flower oral liquid 120mL.
According to the assay method that detects general flavone content in poplar flower oral liquid, detect general flavone content in the present embodiment, the extraction ratio of total flavones is as calculated: 0.188%.According to bis-poplar flower oral liquid quality standard checks of 2010 editions < < Chinese veterinary pharmacopoeia > >, every assay is up to specification.
Claims (4)
1. a preparation method for poplar flower oral liquid, is characterized in that comprising the steps:
(1) flower of poplar is put into extraction pot, add flower of poplar quality 4-6 water doubly, stir and be warming up to 45-50 ℃;
(2) in extraction pot, add the cellulase of flower of poplar quality 3-7%, with the lactic acid solution of 10-50% or the sodium hydroxide solution of 10-50%, regulate pH value to 5.0-5.5, enzymolysis 90-120min at 45-50 ℃;
(3) enzymolysis solution is filtered, collect filtrate, obtain poplar flower oral liquid.
2. the preparation method of poplar flower oral liquid according to claim 1, is characterized in that adding in described step (1) water of 4 times of flower of poplar quality.
3. the preparation method of poplar flower oral liquid according to claim 1, is characterized in that the quality of described step (2) cellulase is 4% of flower of poplar quality.
4. the preparation method of poplar flower oral liquid according to claim 1, is characterized in that in described step (2), enzymolysis time is 120min.
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