CN109157556A - The extracting method and application of dandelion preventing respiratory infection ingredient - Google Patents
The extracting method and application of dandelion preventing respiratory infection ingredient Download PDFInfo
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Abstract
The present invention provides a kind of extraction process of dandelion and applications, including extracting and developing, flocculation, chromatography, drying and other steps, and using the extraction process, the extract yield of dandelion is significantly improved.Dandelion extraction process provided by the invention has the advantages that simple process, safe operation, is suitable for industrialized production；Dandelion extract prepared by the present invention can be used for preparing the drug for the treatment of preventing respiratory infection.
Description
Technical field
The present invention relates to the extraction processes of plant active component, have preventing respiratory infection more specifically to one kind
The plant of effect-dandelion effective component extraction process and its application.
Background technique
Dandelion herbaceos perennial, root is coniform, surface sepia, shrinkage, limb edge have sometimes wave-formed mouth or
Pinniform drastic crack, base portion is gradually narrow at petiole, and often band reddish violet, scape top aubergine are close long by spider's thread shape white for petiole and master pulse
Pubescence；Capitulum, involucre mitriform, achene crineous, long pappus white, 4~October of flowering fruit bearing stage.
Dandelion has highly important medical value, has the function of clearing heat and detoxicating, anti-inflammatory, stomach invigorating, diuresis, dissipating bind, can
Women's cream pain oedema, pharyngitis, acute tonsillitis and all diseases of other heat toxins are treated in smelting.Main active is in dandelion extract
Luteolin, Quercetin, chlorogenic acid and caffeic acid etc..The prior art provides the preparation method of dandelion extract, not
The content of main active in extract on the books, the dandelion extract of active constituent high-content help to realize dandelion
Treat the respiratory tract infection such as acute tonsillitis, pharyngitis.It, should it is therefore desirable to provide a kind of preparation method of dandelion extract
Method and process is simple, is easy to industrial production；More important is dandelion extract is prepared in this way, active constituent is high, can be with
For respiratory tract infection such as therapeutic tonsillitis, pharyngitis.
Summary of the invention
The present invention provides a kind of extraction processes of the high dandelion of simple process, extract yield.
To achieve the goals above, the present invention adopts the following technical scheme:
A kind of extraction process of dandelion, includes the following steps:
1. the preparation of dandelion powder: dandelion impurity elimination being cleaned, is dried, crushes, obtains dandelion powder；
2. enzymatic hydrolysis-Microwave Extraction: 6 ~ 10 times of ethyl alcohol buffers are added, adjust pH value to 4.5 ~ 5.5；Addition enzyme, control temperature 30 ~
It 40 DEG C, digests 0.5 ~ 1.5 hour, then high temperature enzyme deactivation；3 ~ 10min of Microwave Extraction, obtains extracting solution；
3. separation: 2. extracting solution that step is obtained is centrifugated, and obtains supernatant A；
4. optional, flocculation: flocculant being added into step supernatant A 3., stirring 5 ~ 10min of flocculation is centrifugated, collects
Obtain supernatant B；
5. chromatography: 4. supernatant B that 3. supernatant A or step that step obtains obtains passes through macroreticular resin chromatography,
Following elution is successively carried out, (1) is first with distillation 2 ~ 3 column volumes of water elution；(2) 2 ~ 3 cylinders are eluted with 5% ethanol solution
Product；Collect eluent;
6. being dried under reduced pressure, 5. eluent that step obtains is concentrated under reduced pressure, is dried, dandelion extract is obtained.
In the extraction process of above-mentioned dandelion,
Step 1. in, the drying be control 45 ~ 55 DEG C of temperature dried so that water content be not higher than 8%.
Step 1. in, the dandelion powder be smash after, cross 200 meshes, obtain dandelion powder.
Step 2. in, described 6 ~ 10 times of ethyl alcohol buffer, on the basis of dandelion powder, the volume weight of ethyl alcohol buffer
Amount ratio.In some embodiments of the invention, the weight of dandelion powder be 10Kg, step 2. in be added ethyl alcohol buffer body
Product is 60 ~ 100L.
Step 2. in, the ethyl alcohol buffer be pH4.5 ~ 5.5 750 ~ 850ml of buffer and 95% ethyl alcohol 150 ~
The ethyl alcohol buffer (in terms of 1000ml) of 250ml composition, the buffer of pH4.5 ~ 5.5, the preferably buffer of pH5.0；
Disodium hydrogen phosphate-citrate buffer solution of preferred pH4.5 ~ 5.5 of the buffer of pH4.5 ~ 5.5；The phosphorus of more preferable pH5.0
Sour disodium hydrogen-citrate buffer solution.
Step 2. in, the enzyme is the mixture of zytase or zytase and cellulase, preferably zytase
With the mixture of cellulase, more preferable weight ratio is the cellulase of 1:3 ~ 4 and the mixture of zytase, the fiber
Plain enzyme, zytase activity be not less than 100U/g, preferably 200U ~ 500U/g.
Step 2. in, the enzyme, the mass ratio of enzyme and dandelion powder is 0.5 ~ 1.0:100.
Step 2. in, the power of Microwave Extraction is 800 ~ 1000W.
Step 4. in, flocculant is chitosan, and the mass ratio of the flocculant and supernatant A is 0.2 ~ 0.8:100, excellent
It selects flocculant and the mass ratio of supernatant A is 0.3 ~ 0.7:100.
Step 4. in, control pH4.0 ~ 6.0, preferably control pH4.5 ~ 5.5.
Step 4. in, the stirring be 50 ~ 200 turns/min of stirring rate, preferably 100 ~ 150 turns/min.
Step 6. in, the drying, preferably freeze drying.
Dandelion extract extract yield provided by the invention is not less than 2.0%, luteolin in dandelion extract, Mongolian oak
Pi Su, chlorogenic acid and caffeic acid total content are not less than 75%.
The dandelion extract that another aspect of the present invention is to provide the preparation of said extracted method treats preventing respiratory in preparation
Application in infection medicine.
Further, the application, the respiratory tract infection are the respiratory tract infection that pneumonia germ causes.
Further, the application, the respiratory tract infection are the respiratory tract infection that influenza A virus causes
Dandelion powder digests cellulose in dandelion powder under the collective effect of cellulase and xylan, is generating small molecule
While biological active component, the interference of cellulose is reduced, shortens enzymolysis time, reaction condition is mild.Enzymatic hydrolysis and microwave two
Kind extracting mode combines, and can significantly improve extraction (active constituent) efficiency.
It is significantly affected the study found that different buffers have active constituent extract yield in dandelion, disodium hydrogen phosphate-
Citrate buffer solution effect is best.
Compared with prior art, the present invention achieve the utility model has the advantages that
1) present invention feature not high for active constituent extraction efficiency in dandelion powder, takes the work of enzymatic hydrolysis and Microwave Extraction
Skill, so that rate is taken significantly to increase, yield is significantly improved.
2) present invention has been obviously improved extract yield using cellulase and zytase collective effect.
3) present invention eliminates the non-active ingredient impurity in extract by the way of flocculate with chitosan, so that extracting
The content of active constituent is further promoted in object.
4) present invention process is simple, has many advantages, such as energy conservation, is efficient, environmental protection.
Specific embodiment
The invention discloses the infection of dandelion preventing respiratory into the extraction process of leaflet, and those skilled in the art can use for reference
The contents of the present invention are suitably modified realization of process parameters.In particular, it should be pointed out that all similar substitutions and modifications are to ability
It is it will be apparent that they are considered as being included in the scope of the present invention for field technique personnel.Application of the invention has been led to
Preferred embodiment is crossed to be described, related personnel obviously can not depart from the content of present invention, in spirit and scope to this paper institute
The methods and applications stated are modified or appropriate changes and combinations, carry out implementation and application the technology of the present invention.
Next by the following examples the present invention is further explained, but embodiment does not do any restriction to the present invention.
Test method in following embodiments is unless otherwise specified conventional method, original as used in the following examples
Material, reagent material etc. are commercially available products unless otherwise specified.
Extract yield (%): the weight of dandelion powder extracts/dandelion powder weight * 100%
The content (%) of active constituent in extract: luteolin in extract, quercitrin are measured respectively using high performance liquid chromatography
Element, chlorogenic acid and caffeinic content, the content of each component are added to obtain.
The content of active constituent in active constituent total recovery (%)=extract yield * extract
Embodiment 1
1. the preparation of dandelion powder: dandelion impurity elimination being cleaned, 45 DEG C of drying 5h, is crushed, 200 meshes is crossed, obtains dandelion powder
10Kg(water content 7.2%, in terms of dry product, dandelion powder 10Kg)；
2. enzymatic hydrolysis-Microwave Extraction: 60L ethyl alcohol buffer (disodium hydrogen phosphate of pH4.5-citrate buffer solution 45L, 95% second is added
Alcohol 15L), with lemon acid for adjusting pH value to 4.5；Zytase (300U/g) 50g is added, controls 30 DEG C of temperature, enzymatic hydrolysis 1.5 is small
When, then 80 DEG C of enzyme deactivation 3min of high temperature；Microwave Extraction 10min, extraction power are 800W, obtain extracting solution；
3. separation: 2. extracting solution that step is obtained is centrifugated, and obtains supernatant A；
4. chromatography passes through macroreticular resin chromatography to 3. supernatant A that step obtains, successively carries out following elution, (1)
First with distillation 3 column volumes of water elution；(2) 3 column volumes are eluted with 5% ethanol solution；Collect eluent；
5. being dried under reduced pressure, 4. eluent that step obtains is concentrated under reduced pressure, is dried, dandelion extract is obtained.
Embodiment 1 obtains that the extract yield of dandelion extract, active component content, active constituent are always received in extract
Rate result is as follows:
Embodiment 2
1. the preparation of dandelion powder: dandelion impurity elimination being cleaned, 55 DEG C of drying 3h, is crushed, 200 meshes is crossed, obtains dandelion powder
10Kg(water content 6.1%, in terms of dry product, dandelion powder 10Kg)；
2. enzymatic hydrolysis-Microwave Extraction: be added 100L ethyl alcohol buffer (disodium hydrogen phosphate of pH5.5-citrate buffer solution 85L, 95%
Ethyl alcohol 15L), with lemon acid for adjusting pH value to 5.5；Zytase (300U/g) 100g is added, controls 30 DEG C of temperature, enzymatic hydrolysis 0.5
Hour, then 80 DEG C of enzyme deactivation 3min of high temperature；Microwave Extraction 10min, extraction power are 1000W, obtain extracting solution；
3. separation: 2. extracting solution that step is obtained is centrifugated, and obtains supernatant A；
4. chromatography passes through macroreticular resin chromatography to 3. supernatant A that step obtains, successively carries out following elution, (1)
First with distillation 3 column volumes of water elution；(2) 3 column volumes are eluted with 5% ethanol solution；Collect eluent；
5. being dried under reduced pressure, 4. eluent that step obtains is concentrated under reduced pressure, is dried, dandelion extract is obtained.
Embodiment 2 obtains that the extract yield of dandelion extract, active component content, active constituent are always received in extract
Rate result is as follows:
Embodiment 3
1. the preparation of dandelion powder: dandelion impurity elimination being cleaned, 50 DEG C of drying 4h, is crushed, 200 meshes is crossed, obtains dandelion powder
10Kg(water content 6.0%, in terms of dry product, dandelion powder 10Kg)；
2. enzymatic hydrolysis-Microwave Extraction: 80L ethyl alcohol buffer (disodium hydrogen phosphate of pH5.0-citrate buffer solution 64L, 95% second is added
Alcohol 16L), with lemon acid for adjusting pH value to 5.0；Zytase (300U/g) 80g is added, controls 35 DEG C of temperature, enzymatic hydrolysis 1.0 is small
When, then 80 DEG C of enzyme deactivation 3min of high temperature；Microwave Extraction 6min, extraction power are 900W, obtain extracting solution；
3. separation: 2. extracting solution that step is obtained is centrifugated, and obtains supernatant A；
4. chromatography passes through macroreticular resin chromatography to 3. supernatant A that step obtains, successively carries out following elution, (1)
First with distillation 3 column volumes of water elution；(2) 3 column volumes are eluted with 5% ethanol solution；Collect eluent；
5. being dried under reduced pressure, 4. eluent that step obtains is concentrated under reduced pressure, is dried, dandelion extract is obtained.
Embodiment 3 obtains that the extract yield of dandelion extract, active component content, active constituent are always received in extract
Rate result is as follows:
Embodiment 4
1. the preparation of dandelion powder: dandelion impurity elimination being cleaned, 45 DEG C of drying 5h, is crushed, 200 meshes is crossed, obtains dandelion powder
10Kg(water content 7.2%, in terms of dry product, dandelion powder 10Kg)；
2. enzymatic hydrolysis-Microwave Extraction: 60L ethyl alcohol buffer (disodium hydrogen phosphate of pH4.5-citrate buffer solution 45L, 95% second is added
Alcohol 15L), with lemon acid for adjusting pH value to 4.5；Zytase (300U/g) 50g is added, controls 30 DEG C of temperature, enzymatic hydrolysis 1.5 is small
When, then 80 DEG C of enzyme deactivation 3min of high temperature；Microwave Extraction 10min, extraction power are 800W, obtain extracting solution；
3. separation: 2. extracting solution that step is obtained is centrifugated, and obtains supernatant A；
4. flocculation: chitosan is added into step supernatant A 3., additional amount is the 0.3% of supernatant A quality, uses citric acid
PH4.5,100 turns/min stirring flocculation 5min are adjusted, centrifuge separation collects and obtains supernatant B；
5. chromatography passes through macroreticular resin chromatography to 4. supernatant B that step obtains, successively carries out following elution, (1)
First with distillation 2 column volumes of water elution；(2) 2 column volumes are eluted with 5% ethanol solution；Collect eluent；
6. being dried under reduced pressure, 5. eluent that step obtains is concentrated under reduced pressure, is dried, dandelion extract is obtained.
Embodiment 4 obtains the extract yield of dandelion extract, active component content, active constituent total recovery knot in extract
Fruit is as follows:
Embodiment 5
1. the preparation of dandelion powder: dandelion impurity elimination being cleaned, 55 DEG C of drying 3h, is crushed, 200 meshes is crossed, obtains dandelion powder
10Kg(water content 6.1%, in terms of dry product, dandelion powder 10Kg)；
2. enzymatic hydrolysis-Microwave Extraction: be added 100L ethyl alcohol buffer (disodium hydrogen phosphate of pH5.5-citrate buffer solution 85L, 95%
Ethyl alcohol 15L), with lemon acid for adjusting pH value to 5.5；Zytase (300U/g) 100g is added, controls 30 DEG C of temperature, enzymatic hydrolysis 0.5
Hour, then 80 DEG C of enzyme deactivation 3min of high temperature；Microwave Extraction 10min, extraction power are 1000W, obtain extracting solution；
3. separation: 2. extracting solution that step is obtained is centrifugated, and obtains supernatant A；
4. flocculation: chitosan is added into step supernatant A 3., additional amount is the 0.6% of supernatant A quality, uses citric acid
PH5.5,150 turns/min stirring flocculation 10min are adjusted, centrifuge separation collects and obtains supernatant B；
5. chromatography passes through macroreticular resin chromatography to 4. supernatant B that step obtains, successively carries out following elution,
(1) first with distillation 2 column volumes of water elution；(2) 2 column volumes are eluted with 5% ethanol solution；Collect eluent；
6. being dried under reduced pressure, 5. eluent that step obtains is concentrated under reduced pressure, is dried, dandelion extract is obtained.
Embodiment 5 obtains that the extract yield of dandelion extract, active component content, active constituent are always received in extract
Rate result is as follows:
Embodiment 6
1. the preparation of dandelion powder: dandelion impurity elimination being cleaned, 50 DEG C of drying 4h, is crushed, 200 meshes is crossed, obtains dandelion powder
10Kg(water content 6.0%, in terms of dry product, dandelion powder 10Kg)；
2. enzymatic hydrolysis-Microwave Extraction: 80L ethyl alcohol buffer (disodium hydrogen phosphate of pH5.0-citrate buffer solution 64L, 95% second is added
Alcohol 16L), with lemon acid for adjusting pH value to 5.0；Zytase (300U/g) 80g is added, controls 35 DEG C of temperature, enzymatic hydrolysis 1.0 is small
When, then 80 DEG C of enzyme deactivation 3min of high temperature；Microwave Extraction 6min, extraction power are 900W, obtain extracting solution；
3. separation: 2. extracting solution that step is obtained is centrifugated, and obtains supernatant A；
4. flocculation: chitosan is added into step supernatant A 3., additional amount is the 0.4% of supernatant A quality, uses citric acid
PH5.0,120 turns/min stirring flocculation 8min are adjusted, centrifuge separation collects and obtains supernatant B；
5. chromatography passes through macroreticular resin chromatography to 4. supernatant B that step obtains, successively carries out following elution, (1)
First with distillation 2 column volumes of water elution；(2) 2 column volumes are eluted with 5% ethanol solution；Collect eluent;
6. being dried under reduced pressure, 5. eluent that step obtains is concentrated under reduced pressure, is dried, dandelion extract is obtained.
Embodiment 6 obtains that the extract yield of dandelion extract, active component content, active constituent are always received in extract
Rate result is as follows:
Comparative analysis embodiment 1-3 and embodiment 4-6, can significantly find out: 4. step can be obviously improved extraction
The content of active constituent in object.Specifically it see the table below:
Embodiment 7
1. the preparation of dandelion powder: dandelion impurity elimination being cleaned, 50 DEG C of drying 4h, is crushed, 200 meshes is crossed, obtains dandelion powder
10Kg(water content 6.0%, in terms of dry product, dandelion powder 10Kg)；
2. enzymatic hydrolysis-Microwave Extraction: 90L ethyl alcohol buffer (disodium hydrogen phosphate of pH5.5-citrate buffer solution 70L, 95% second is added
Alcohol 20L), with lemon acid for adjusting pH value to 5.2；Zytase (300U/g) 90g is added, controls 35 DEG C of temperature, enzymatic hydrolysis 1.0 is small
When, then 80 DEG C of enzyme deactivation 3min of high temperature；Microwave Extraction 6min, extraction power are 900W, obtain extracting solution；
3. separation: 2. extracting solution that step is obtained is centrifugated, and obtains supernatant A；
4. flocculation: chitosan is added into step supernatant A 3., additional amount is the 0.5% of supernatant A quality, uses citric acid
PH5.0,130 turns/min stirring flocculation 7min are adjusted, centrifuge separation collects and obtains supernatant B；
5. chromatography passes through macroreticular resin chromatography to 4. supernatant B that step obtains, successively carries out following elution, (1)
First with distillation 2 column volumes of water elution；(2) 2 column volumes are eluted with 5% ethanol solution；Collect eluent;
6. being dried under reduced pressure, 5. eluent that step obtains is concentrated under reduced pressure, is dried, dandelion extract is obtained.
Embodiment 7 obtains that the extract yield of dandelion extract, active component content, active constituent are always received in extract
Rate result is as follows:
Embodiment 8
1. the preparation of dandelion powder: dandelion impurity elimination being cleaned, 50 DEG C of drying 4h, is crushed, 200 meshes is crossed, obtains dandelion powder
10Kg(water content 6.0%, in terms of dry product, dandelion powder 10Kg)；
2. enzymatic hydrolysis-Microwave Extraction: 80L ethyl alcohol buffer (disodium hydrogen phosphate of pH5.0-citrate buffer solution 64L, 95% second is added
Alcohol 16L), with lemon acid for adjusting pH value to 5.0；Cellulase (200U/g) 20g, zytase (300U/g) 60g, control is added
It 35 DEG C of temperature, digests 1.0 hours, then 80 DEG C of enzyme deactivation 3min of high temperature；Microwave Extraction 6min, extraction power are 900W, are mentioned
Take liquid；
3. separation: 2. extracting solution that step is obtained is centrifugated, and obtains supernatant A；
4. flocculation: chitosan is added into step supernatant A 3., additional amount is the 0.4% of supernatant A quality, uses citric acid
PH5.0,120 turns/min stirring flocculation 8min are adjusted, centrifuge separation collects and obtains supernatant B；
5. chromatography passes through macroreticular resin chromatography to 4. supernatant B that step obtains, successively carries out following elution, (1)
First with distillation 2 column volumes of water elution；(2) 2 column volumes are eluted with 5% ethanol solution；Collect eluent;
6. being dried under reduced pressure, 5. eluent that step obtains is concentrated under reduced pressure, is dried, dandelion extract is obtained.
Embodiment 8 obtains that the extract yield of dandelion extract, active component content, active constituent are always received in extract
Rate result is as follows:
Embodiment 9
1. the preparation of dandelion powder: dandelion impurity elimination being cleaned, 50 DEG C of drying 4h, is crushed, 200 meshes is crossed, obtains dandelion powder
10Kg(water content 6.0%, in terms of dry product, dandelion powder 10Kg)；
2. enzymatic hydrolysis-Microwave Extraction: 80L ethyl alcohol buffer (disodium hydrogen phosphate of pH5.0-citrate buffer solution 64L, 95% second is added
Alcohol 16L), with lemon acid for adjusting pH value to 5.0；Cellulase (200U/g) 16g, zytase (300U/g) 64g, control is added
It 35 DEG C of temperature, digests 1.0 hours, then 80 DEG C of enzyme deactivation 3min of high temperature；Microwave Extraction 6min, extraction power are 900W, are mentioned
Take liquid；
3. separation: 2. extracting solution that step is obtained is centrifugated, and obtains supernatant A；
4. flocculation: chitosan is added into step supernatant A 3., additional amount is the 0.4% of supernatant A quality, uses citric acid
PH5.0,120 turns/min stirring flocculation 8min are adjusted, centrifuge separation collects and obtains supernatant B；
5. chromatography passes through macroreticular resin chromatography to 4. supernatant B that step obtains, successively carries out following elution, (1)
First with distillation 2 column volumes of water elution；(2) 2 column volumes are eluted with 5% ethanol solution；Collect eluent;
6. being dried under reduced pressure, 5. eluent that step obtains is concentrated under reduced pressure, is dried, dandelion extract is obtained.
Embodiment 9 obtains that the extract yield of dandelion extract, active component content, active constituent are always received in extract
Rate result is as follows:
Embodiment 10
1. the preparation of dandelion powder: dandelion impurity elimination being cleaned, 50 DEG C of drying 4h, is crushed, 200 meshes is crossed, obtains dandelion powder
10Kg(water content 6.0%, in terms of dry product, dandelion powder 10Kg)；
2. enzymatic hydrolysis-Microwave Extraction: 80L ethyl alcohol buffer (disodium hydrogen phosphate of pH5.0-citrate buffer solution 64L, 95% second is added
Alcohol 16L), with lemon acid for adjusting pH value to 5.0；Cellulase (200U/g) 18g, zytase (300U/g) 62g, control is added
It 35 DEG C of temperature, digests 1.0 hours, then 80 DEG C of enzyme deactivation 3min of high temperature；Microwave Extraction 6min, extraction power are 900W, are mentioned
Take liquid；
3. separation: 2. extracting solution that step is obtained is centrifugated, and obtains supernatant A；
4. flocculation: chitosan is added into step supernatant A 3., additional amount is the 0.4% of supernatant A quality, uses citric acid
PH5.0,120 turns/min stirring flocculation 8min are adjusted, centrifuge separation collects and obtains supernatant B；
5. chromatography passes through macroreticular resin chromatography to 4. supernatant B that step obtains, successively carries out following elution, (1)
First with distillation 2 column volumes of water elution；(2) 2 column volumes are eluted with 5% ethanol solution；Collect eluent;
6. being dried under reduced pressure, 5. eluent that step obtains is concentrated under reduced pressure, is dried, dandelion extract is obtained.
Embodiment 10 obtains that the extract yield of dandelion extract, active component content, active constituent are total in extract
Yield result is as follows:
Embodiment 11
1. the preparation of dandelion powder: dandelion impurity elimination being cleaned, 50 DEG C of drying 4h, is crushed, 200 meshes is crossed, obtains dandelion powder
10Kg(water content 6.0%, in terms of dry product, dandelion powder 10Kg)；
2. enzymatic hydrolysis-Microwave Extraction: 80L ethyl alcohol buffer is added, adjusting pH value to 5.0(ethyl alcohol buffer see the table below)；It is added fine
Element enzyme (200U/g) 18g, zytase (300U/g) 62g are tieed up, controls 35 DEG C of temperature, digests 1.0 hours, then goes out for 80 DEG C of high temperature
Enzyme 3min；Microwave Extraction 6min, extraction power are 900W, obtain extracting solution；
3. separation: 2. extracting solution that step is obtained is centrifugated, and obtains supernatant A；
4. flocculation: chitosan is added into step supernatant A 3., additional amount is the 0.4% of supernatant A quality, uses citric acid
PH5.0,120 turns/min stirring flocculation 8min are adjusted, centrifuge separation collects and obtains supernatant B；
5. chromatography passes through macroreticular resin chromatography to 4. supernatant B that step obtains, successively carries out following elution, (1)
First with distillation 2 column volumes of water elution；(2) 2 column volumes are eluted with 5% ethanol solution；Collect eluent;
6. being dried under reduced pressure, 5. eluent that step obtains is concentrated under reduced pressure, is dried, dandelion extract is obtained.
Embodiment 11 obtains that the extract yield of dandelion extract, active component content, active constituent are total in extract
Yield result is as follows:
Comparative analysis embodiment 11-1 to embodiment 11-8 and embodiment 10, specifically see the table below:
It can be seen that
1. comparative analysis embodiment 11-1, embodiment 11-2, embodiment 11-3, the pH of buffer influence the yield of extract and mention
The content for taking active constituent in object, when use pH is 4.5 ~ 5.5 acetic acid-sodium acetate buffer solution, the acetate buffer that pH is 5.0
Liquid extraction effect is more preferable；
2. the pH of comparative analysis embodiment 11-4, embodiment 11-5, embodiment 10, buffer influences yield and the extraction of extract
The content of active constituent in object, when use pH is 4.5 ~ 5.5 disodium hydrogen phosphates-citrate buffer solution, the phosphoric acid hydrogen two that pH is 5.0
Sodium-citrate buffer solution extraction effect is more preferable；
3. comparative analysis embodiment 11-6, embodiment 11-7, embodiment 11-8, the pH of buffer influence the yield of extract and mention
The content for taking active constituent in object, when use pH is 4.5 ~ 5.5 disodium hydrogen phosphates-potassium phosphate buffer, the phosphorus that pH is 5.0
Sour disodium hydrogen-potassium phosphate buffer extraction effect is more preferable；
4. comparative analysis embodiment 11-1, embodiment 11-4, embodiment 11-6, the different buffers that pH value is 4.5 influence shadow
The content of active constituent in the yield and extract of extract is rung, the disodium hydrogen phosphate that pH is 4.5-citrate buffer solution extracts effect
Fruit is more preferable；
5. comparative analysis embodiment 11-2, embodiment 11-5, embodiment 11-7, the different buffers that pH value is 5.5 influence shadow
The content of active constituent in the yield and extract of extract is rung, the disodium hydrogen phosphate that pH is 5.5-citrate buffer solution extracts effect
Fruit is more preferable；
6. comparative analysis embodiment 11-3, embodiment 10, embodiment 11-8, the different buffers that pH value is 5.0 influence
The content of active constituent in the yield and extract of extract, disodium hydrogen phosphate-citrate buffer solution extraction effect that pH is 5.0
More preferably；
Comprehensive analysis 1., 2., 3., step 2. used in pH4.5 ~ 5.5 buffer, the preferred buffer of pH5.0；Further
, comprehensive analysis 4., 5., 6., step 2. used in pH4.5 ~ 5.5 buffer, the preferred phosphoric acid hydrogen two of pH4.5 ~ 5.5
Sodium-citrate buffer solution；Disodium hydrogen phosphate-citrate buffer solution of more preferable pH5.0.
Embodiment 12
1. the preparation of dandelion powder: dandelion impurity elimination being cleaned, 50 DEG C of drying 4h, is crushed, 200 meshes is crossed, obtains dandelion powder
10Kg(water content 6.0%, in terms of dry product, dandelion powder 10Kg)；
2. enzymatic hydrolysis-Microwave Extraction: 80L ethyl alcohol buffer (disodium hydrogen phosphate of pH5.0-citrate buffer solution 64L, 95% second is added
Alcohol 16L), with lemon acid for adjusting pH value to 5.0；The specific enzyme of enzyme 80g(, which is added, see the table below), 35 DEG C of temperature are controlled, enzymatic hydrolysis 1.0 is small
When, then 80 DEG C of enzyme deactivation 3min of high temperature；Microwave Extraction 6min, extraction power are 900W, obtain extracting solution；
3. separation: 2. extracting solution that step is obtained is centrifugated, and obtains supernatant A；
4. flocculation: chitosan being added into step supernatant A 3., additional amount is the 0.4% of supernatant A quality, with citric acid tune
PH5.5,120 turns/min stirring flocculation 8min are saved, centrifuge separation collects and obtains supernatant B；
5. chromatography passes through macroreticular resin chromatography to 4. supernatant B that step obtains, successively carries out following elution, (1)
First with distillation 2 column volumes of water elution；(2) 2 column volumes are eluted with 5% ethanol solution；Collect eluent;
6. being dried under reduced pressure, 5. eluent that step obtains is concentrated under reduced pressure, is dried, dandelion extract is obtained.
Embodiment 12 obtains that the extract yield of dandelion extract, active component content, active constituent are total in extract
Yield result is as follows:
Embodiment 13
1. the preparation of dandelion powder: dandelion impurity elimination being cleaned, 50 DEG C of drying 4h, is crushed, 200 meshes is crossed, obtains dandelion powder
10Kg(water content 6.0%, in terms of dry product, dandelion powder 10Kg)；
2. enzymatic hydrolysis-Microwave Extraction: 80L ethyl alcohol buffer (disodium hydrogen phosphate of pH5.0-citrate buffer solution 64L, 95% second is added
Alcohol 16L), with lemon acid for adjusting pH value to 5.0；Zytase (300U/g) 80g is added, controls 35 DEG C of temperature, enzymatic hydrolysis 1.0 is small
When, then 80 DEG C of enzyme deactivation 3min of high temperature；Microwave Extraction 6min, extraction power are 900W, obtain extracting solution；
3. separation: 2. extracting solution that step is obtained is centrifugated, and obtains supernatant A；；
4. flocculation: chitosan is added into step supernatant A 3., adjusts pH, stirring flocculation (specific process parameter see the table below),
Centrifuge separation, collection obtain supernatant B；
5. chromatography passes through macroreticular resin chromatography to 4. supernatant B that step obtains, successively carries out following elution, (1)
First with distillation 2 column volumes of water elution；(2) 2 column volumes are eluted with 5% ethanol solution；Collect eluent;
6. being dried under reduced pressure, 5. eluent that step obtains is concentrated under reduced pressure, is dried, dandelion extract is obtained.
Carrying out turbidity to 4. supernatant B that step obtains, (measuring method is 2015 editions three clarity inspections of Chinese Pharmacopoeia
Method) compare, as a result it see the table below:
It applies example 13 and obtains that the extract yield of dandelion extract, active component content, active constituent are always received in extract
Rate result is as follows:
Comprehensive analysis embodiment 13-1 ~ embodiment 13-6 and embodiment 6, step 4. in flocculant additional amount, flocculated
PH value and flocculated stirring rate etc. influence flocculating effect, influence the turbidity of supernatant B, further influence the yield of extract
And in extract active constituent content.
1. the additional amount of comprehensive analysis embodiment 13-1 ~ embodiment 13-4 and embodiment 6, flocculant influences flocculating effect,
Additional amount is 0.2 ~ 0.8%, and the amount of being preferably added to is 0.3% ~ 0.7%；
2. comprehensive analysis embodiment 13-5 ~ embodiment 13-8 and embodiment 6, flocculated pH value influences flocculating effect, and flocculation pH is
4.0 ~ 6.0, preferably pH are 4.5 ~ 5.5；
3. comprehensive analysis embodiment 13-9 ~ embodiment 13-12 and embodiment 6, flocculated stirring rate influences flocculating effect, flocculation
Stirring rate be 50 turns/min ~ 200 turn/min, preferably flocculated stirring rate be 100 turns/min ~ 150 turn/min.
Comparative example 1
1. the preparation of dandelion powder: dandelion impurity elimination being cleaned, 50 DEG C of drying 4h, is crushed, 200 meshes is crossed, obtains dandelion powder
10Kg(water content 6.0%, in terms of dry product, dandelion powder 10Kg)；
2. enzymatic hydrolysis-Microwave Extraction: being added 80L ethyl alcohol buffer (composition and ratio see the table below), with lemon acid for adjusting pH value to 5.0；
Cellulase (200U/g) 18g, zytase (300U/g) 62g is added, controls 35 DEG C of temperature, digests 1.0 hours, then high temperature
80 DEG C of enzyme deactivation 3min；Microwave Extraction 6min, extraction power are 900W, obtain extracting solution；
3. separation: 2. extracting solution that step is obtained is centrifugated, and obtains supernatant A；
4. flocculation: chitosan is added into step supernatant A 3., additional amount is the 0.4% of supernatant A quality, uses citric acid
PH5.5,120 turns/min stirring flocculation 8min are adjusted, centrifuge separation collects and obtains supernatant B；
5. chromatography passes through macroreticular resin chromatography to 4. supernatant B that step obtains, successively carries out following elution, (1)
First with distillation 2 column volumes of water elution；(2) 2 column volumes are eluted with 5% ethanol solution；Collect eluent;
6. being dried under reduced pressure, 5. eluent that step obtains is concentrated under reduced pressure, is dried, dandelion extract is obtained.
Comparative example 1 obtains that the extract yield of dandelion extract, active component content, active constituent are always received in extract
Rate result is as follows
Analyze comparative example 1, it will thus be seen that the composition of ethyl alcohol buffer influences active constituent in the yield and extract of extract
Content.
Comparative example 2:
1. the preparation of dandelion powder: dandelion impurity elimination being cleaned, 50 DEG C of drying 4h, is crushed, 200 meshes is crossed, obtains dandelion powder
10Kg(water content 6.0%, in terms of dry product, dandelion powder 10Kg)；
2. enzymatic hydrolysis-Microwave Extraction: 80L ethyl alcohol buffer is added, with lemon acid for adjusting pH value (details see the table below)；It is added fine
Element enzyme (200U/g) 18g, zytase (300U/g) 62g are tieed up, controls 35 DEG C of temperature, digests 1.0 hours, then goes out for 80 DEG C of high temperature
Enzyme 3min；Microwave Extraction 6min, extraction power are 900W, obtain extracting solution；
3. separation: 2. extracting solution that step is obtained is centrifugated, and obtains supernatant A；
4. flocculation: chitosan is added into step supernatant A 3., additional amount is the 0.4% of supernatant A quality, uses citric acid
PH5.5,120 turns/min stirring flocculation 8min are adjusted, centrifuge separation collects and obtains supernatant B；
5. chromatography passes through macroreticular resin chromatography to 4. supernatant B that step obtains, successively carries out following elution, (1)
First with distillation 2 column volumes of water elution；(2) 2 column volumes are eluted with 5% ethanol solution；Collect eluent;
6. being dried under reduced pressure, 5. eluent that step obtains is concentrated under reduced pressure, is dried, dandelion extract is obtained.
Comparative example 2 obtains that the extract yield of dandelion extract, active component content, active constituent are always received in extract
Rate result is as follows
Analyze comparative example 2, it will thus be seen that active constituent contains in the yield and extract of the pH influence extract of ethyl alcohol buffer
Amount, the buffer extraction effect of pH value 4.0,6.0,7.0 are poor.
Comparative example 3
1. the preparation of dandelion powder: dandelion impurity elimination being cleaned, 50 DEG C of drying 4h, is crushed, 200 meshes is crossed, obtains dandelion powder
10Kg(water content 6.0%, in terms of dry product, dandelion powder 10Kg)；
2. enzymatic hydrolysis-Microwave Extraction: 80L ethyl alcohol buffer (disodium hydrogen phosphate of pH5.0-citrate buffer solution 64L, 95% second is added
Alcohol 16L), with lemon acid for adjusting pH value to 5.0；Cellulase (200U/g) 80g is added, controls 35 DEG C of temperature, enzymatic hydrolysis 1.0 is small
When, then 80 DEG C of enzyme deactivation 3min of high temperature；Microwave Extraction 6min, extraction power are 900W, obtain extracting solution；
3. separation: 2. extracting solution that step is obtained is centrifugated, and obtains supernatant A；
4. flocculation: chitosan is added into step supernatant A 3., additional amount is the 0.4% of supernatant A quality, uses citric acid
PH5.5,120 turns/min stirring flocculation 8min are adjusted, centrifuge separation collects and obtains supernatant B；
5. chromatography passes through macroreticular resin chromatography to 4. supernatant B that step obtains, successively carries out following elution, (1)
First with distillation 2 column volumes of water elution；(2) 2 column volumes are eluted with 5% ethanol solution；Collect eluent;
6. being dried under reduced pressure, 5. eluent that step obtains is concentrated under reduced pressure, is dried, dandelion extract is obtained；
Comparative example 3 obtains the extract yield of dandelion extract, active component content, active constituent total recovery knot in extract
Fruit is as follows:
Comparative analysis embodiment 6, embodiment 8-10, embodiment 12 and comparative example 3, it will thus be seen that
1. from comparative example 3 it can be seen that cellulase is used alone, extract yield is lower than 2.0%, active constituent in extract
Content is also not achieved 75%；
2. comparative analysis comparative example 3 and embodiment 6, it will thus be seen that compared with cellulase, be obviously improved and mentioned using zytase
Take the content of active constituent in object yield and extract, extract yield is promoted to 3.0% by 1.5%, 100% is promoted, in extract
The content of active constituent is promoted to 82.8% by 72.5%, promotes 14.2%.
3. comparative analysis embodiment 12-1, embodiment 12-2 and embodiment 6, it will thus be seen that with exclusive use zytase
It compares, using the mixture of zytase and cellulase, the content of active constituent in extract can be promoted, promoted by 82.8%
To 85.7%, 3.5% is promoted.
4. comparative analysis embodiment 8-10 and embodiment 6, it will thus be seen that compared with zytase is used alone, weight ratio
For the cellulase of 1:3 ~ 4 and the mixture of zytase, containing for active constituent in the yield and extract of extract can be promoted
Amount, extract yield are promoted to 3.6%(embodiment 8-10 average value by 3.0%), 20% is promoted, the middle active constituent of extract
Content is promoted to 86.3%(embodiment 8-10 average value by 82.8%), promote 4.2%.
5. comprehensive analysis embodiment 8-10, embodiment 12-1 and embodiment 12-2, when 2. enzyme zytase that step uses
With the mixture of cellulase, weight ratio be 1:3 ~ 4 cellulase and zytase mixture effect it is more preferable.
Embodiment 14: application of the dandelion extract in preventing respiratory infection medicine
1, experimental animal:: ICR mouse, 13~16g of weight, half male and half female；
2, the preparation for the respiratory tract infection animal model that pneumonia germ causes:
Model group: being anesthetized with ether makes mouse slightly go into a coma, fixation of lying on the back；0.05 mL of pneumonia bacterium solution is taken with 1 mL syringe, is used
Hand touches mouse web portion, touches between the rib cage of the 3rd~4, left side；Inserting needle passes through thoracic cavity, is pierced into lung and carefully injects.Injection is completed
Afterwards, mouse is placed in cage and observes 4 h, without obvious different shape, then model success.
Normal group: ICR mouse 12, isometric physiological saline is injected intraperitoneally
3, grouping and administration
Grouping and administration: model group mouse 84, it is divided into 7 groups, every group 12.
(1) Normal group: same amount of normal saline stomach-filling.
(2) the respiratory tract infection model group that pneumonia germ causes: model group 12, same amount of normal saline stomach-filling；
(3) dandelion extract group prepared by embodiment 1: model group 12, dosage gives 500mg/ with active ingredient daily
Kg stomach-filling, gives in three times.
(4) dandelion extract group prepared by embodiment 3: model group 12, dosage is given daily with active ingredient
500mg/Kg stomach-filling, gives in three times.
(5) dandelion extract group prepared by embodiment 4: model group 12, dosage is given daily with active ingredient
500mg/Kg stomach-filling, gives in three times.
(6) dandelion extract group prepared by embodiment 6: model group 12, dosage is given daily with active ingredient
500mg/Kg stomach-filling, gives in three times.
(7) dandelion extract group prepared by embodiment 8: model group 12, dosage is given daily with active ingredient
500mg/Kg stomach-filling, gives in three times.
(8) dandelion extract group prepared by embodiment 10: model group 12, dosage is given daily with active ingredient
500mg/Kg stomach-filling, gives in three times.
Animal ad lib, drinking-water during experiment.
It is evaluated after being administered 2 weeks
4, result
After administration, observation mouse physiology, activity condition are simultaneously recorded, 2 times a day.Normal group mouse spirit is good, and action is flexibly quick
Victory, hair luster, breathing is normal, and diet is normal, weight natural increase；Model group mouse after pneumonia infection germ the 1st,
Performance in 2 days is short of breath, and activity is decreased obviously, loss of appetite etc., and dandelion extract group had part small since the 3rd day
Mouse improves, and occurs significant improvement after 7 days, and physiology, activity condition are normal after 10 days, but model control group is dead
It is more serious to die situation
From embodiment 14 it can be seen that
1. dandelion extract of the invention has good therapeutic effect to the respiratory tract infection that pneumonia causes, can reduce at least
50% death rate.
2. the content of active constituent affects therapeutic effect in extract, when active component content is higher than 85%, treatment effect
Fruit is more preferable, it is possible to reduce 75% death rate.
Embodiment 15: application of the dandelion extract in preventing respiratory infection medicine
1, experimental animal: ICR mouse, 13~16g of weight, half male and half female；
2, the preparation for the respiratory tract infection animal model that influenza A virus causes:
Model group: being anesthetized with ether makes mouse slightly go into a coma, fixation of lying on the back；With 4LD50The infection of influenza A virus drop nose, often
Mouse 30 μ l, infective dose 1LD50 , model successfully.
Normal group: ICR mouse 12, with physiological saline collunarium, every 30 μ l of mouse.
3, grouping and administration
Grouping and administration: model group mouse 84, it is divided into 7 groups, every group 12.
(1) Normal group: same amount of normal saline stomach-filling.
(2) the respiratory tract infection model group that influenza A virus causes: model group 12, same amount of normal saline stomach-filling；
(3) dandelion extract group prepared by embodiment 2: model group 12, dosage gives 500mg/ with active ingredient daily
Kg stomach-filling, gives in three times.
(4) dandelion extract group prepared by embodiment 5: model group 12, dosage is given daily with active ingredient
600mg/Kg stomach-filling, gives in three times.
(5) dandelion extract group prepared by embodiment 6: model group 12, dosage is given daily with active ingredient
600mg/Kg stomach-filling, gives in three times.
(6) dandelion extract group prepared by embodiment 7: model group 12, dosage is given daily with active ingredient
600mg/Kg stomach-filling, gives in three times.
(7) dandelion extract group prepared by embodiment 9: model group 12, dosage is given daily with active ingredient
600mg/Kg stomach-filling, gives in three times.
(8) dandelion extract group prepared by embodiment 10: model group 12, dosage is given daily with active ingredient
600mg/Kg stomach-filling, gives in three times.
Animal ad lib, drinking-water during experiment.
It is evaluated after being administered 2 weeks
4, result
After administration, observation mouse physiology, activity condition are simultaneously recorded, 2 times a day.Normal group mouse spirit is good, and action is flexibly quick
Victory, hair luster, breathing is normal, and diet is normal, weight natural increase；Do not occur in 2 days after model group mouse infection virus
Obvious abnormal symptom, the 3rd day starts to be short of breath, and is slow in action, and hair is matt, the performance such as loss of appetite, syntexis；Pu
Public English extract group symptom is substantially reduced, and physiology, activity condition are normal after 10 days, but model control group death condition is more tight
Weight.
From embodiment 15 it can be seen that
1. dandelion extract of the invention, which causes respiratory tract infection to influenza A virus, good therapeutic effect, can drop
Low at least 41.7% death rate.
2. the content of active constituent affects therapeutic effect in extract, when active component content is higher than 85%, treatment effect
Fruit is more preferable, it is possible to reduce 66.7% death rate.
Claims (10)
1. a kind of extraction process of dandelion, includes the following steps:
The preparation of dandelion powder: dandelion impurity elimination is cleaned, is dried, crushes, obtains dandelion powder；
Enzymatic hydrolysis-Microwave Extraction: being added 6 ~ 10 times of ethyl alcohol buffers, adjusts pH value to 4.5 ~ 5.5；Enzyme is added, controls temperature 30 ~ 40
DEG C, it digests 0.5 ~ 1.5 hour, then high temperature enzyme deactivation；3 ~ 10min of Microwave Extraction, obtains extracting solution；
Separation: 2. extracting solution that step is obtained is centrifugated, and obtains supernatant A；
It is optional, flocculation: flocculant, stirring 5 ~ 10min of flocculation is added into step supernatant A 3., centrifuge separation is collected
To supernatant B；
Chromatography: 4. supernatant B that 3. supernatant A or step that step obtains obtains passes through macroreticular resin chromatography, according to
It is eluted below secondary progress, (1) is first with distillation 2 ~ 3 column volumes of water elution；(2) 2 ~ 3 column volumes are eluted with 5% ethanol solution；
Collect eluent;
It is dried under reduced pressure, 5. eluent that step obtains is concentrated under reduced pressure, is dried, dandelion extract is obtained；
In above-mentioned taking technique,
The step 1. in, the drying be control 45 ~ 55 DEG C of temperature dried so that water content be not higher than 8%；
The step 1. in, the dandelion powder be smash after, cross 200 meshes, obtain dandelion powder；
The step 2. in, the ethyl alcohol buffer be pH4.5 ~ 5.5 750 ~ 850ml of buffer and 95% ethyl alcohol 150 ~
The 1000ml ethyl alcohol buffer of 250ml composition；
The step 2. in, the enzyme is zytase or zytase and cellulase moral mixture；
The step 2. in, the enzyme, the mass ratio of enzyme and dandelion powder is 0.5 ~ 1.0:100；
The step 2. in, the power of Microwave Extraction is 800 ~ 1000W.
2. extraction process as described in claim 1, which is characterized in that step 2. in, the buffer of pH4.5 ~ 5.5,
It is preferred that the buffer of pH5.0.
3. extraction process as described in claim 1, which is characterized in that step 2. in, the buffer of pH4.5 ~ 5.5 is excellent
Select disodium hydrogen phosphate-citrate buffer solution of pH4.5 ~ 5.5；Disodium hydrogen phosphate-citrate buffer solution of more preferable pH5.0.
4. extraction process as described in claim 1, which is characterized in that step 2. in, the enzyme, preferred weight ratio be 1:3 ~
4 cellulase and the mixture of zytase.
5. extraction process as described in claim 1, which is characterized in that step 4. in, flocculant is chitosan, the flocculant
Mass ratio with supernatant A is 0.2 ~ 0.8:100；The stirring is 50 ~ 200 turns/min of stirring rate.
6. extraction process as claimed in claim 5, which is characterized in that step 4. in, the quality of the flocculant and supernatant A
Ratio is 0.3 ~ 0.7:100；Control pH4.0 ~ 6.0, to control pH4.5 ~ 5.5.
7. extraction process as claimed in claim 5, which is characterized in that step 4. in, the stirring be stirring rate 100 ~
150 turns/min.
8. application of any dandelion extract of claim 1-7 preparation in preparation treatment preventing respiratory infection medicine.
9. application as claimed in claim 9, which is characterized in that the respiratory tract infection is the respiratory tract that pneumonia germ causes
Infection.
10. application as claimed in claim 9, which is characterized in that the respiratory tract infection is what influenza A virus caused
Respiratory tract infection.
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