CN104997826A - Response surface optimization extraction and purification method of highly-pure crowndaisy chrysanthemum total flavonoids - Google Patents
Response surface optimization extraction and purification method of highly-pure crowndaisy chrysanthemum total flavonoids Download PDFInfo
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Abstract
The invention relates to a response surface optimization extraction and purification method of highly-pure crowndaisy chrysanthemum total flavonoids. The method comprises the following steps: carrying out crude extraction on crowndaisy chrysanthemum total flavonoids through an ultrasonic assisting technology, separating the crowndaisy chrysanthemum total flavonoids through a double water phase extraction technology, and separating and purifying the crowndaisy chrysanthemum total flavonoids through macro-porous resin to optimize in order to obtain the crowndaisy chrysanthemum total flavonoids with high content. Ultrasonic assisted extraction and double water phase extraction are carried out on crowndaisy chrysanthemum, so the extraction rate of the crowndaisy chrysanthemum total flavonoids is high, and the extraction rate of other non-flavone components is low; and macro-porous resin is adopted to separate and purify, so the method has the characteristics of simple operation process, low cost and easy operation.
Description
Technical field
The present invention relates to a kind of effective ingredients in plant extraction process, be specifically related to the extraction and purification process of preferred high-purity Caulis et Folium Chrysanthemi segeti total flavones of a kind of response surface.
Background technology
Caulis et Folium Chrysanthemi segeti, it is the stem and leaf of feverfew Chrysanthemum carinatum Schousb. and Caulis et Folium Chrysanthemi segeti, because its flower is the spitting image of Herba Dendranthematis indici, so have another name called Flos Chrysanthemi dish, the stem of Caulis et Folium Chrysanthemi segeti and leaf can with foods, the clearing heat in QI system of Artemisia, the sweet perfume (or spice) of chrysanthemum, general nutrient components is off guard without institute, especially the content of carotene exceedes general vegetable, and the traditional Chinese medical science thinks that Caulis et Folium Chrysanthemi segeti is pungent, sweet, flat, returns spleen, stomach warp, energy whetting appetite, blood pressure lowering supplementing the brain etc., there is certain curative effect, the effect cleared away heart-fire of nourishing blood in addition to cough ant phlegm, incoordination between the spleen and stomach, constipation, hypomnesis, the disease such as not have enough sleep, be relatively applicable to coronary heart disease, hypertensive patient eats.Flavonoid substances has anticancer, antiviral, the multiple physiologically active pharmacologic actions such as immunity moderation power, treatment cardiovascular and cerebrovascular disease, osteoporosis and diabetic complication.Have containing comparatively abundant flavone in research report Caulis et Folium Chrysanthemi segeti, Caulis et Folium Chrysanthemi segeti total flavones possesses good scavenging action to hydroxy radical.But the purity of the Caulis et Folium Chrysanthemi segeti total flavones that current extracting method obtains is generally on the low side, governs the research of Caulis et Folium Chrysanthemi segeti total flavones pharmacology and quality control always.Therefore invent a kind of extraction in Caulis et Folium Chrysanthemi segeti to obtain the high Caulis et Folium Chrysanthemi segeti total flavones of purity content and become particularly important.
Summary of the invention
The object of the invention is to the defect overcoming prior art, a kind of extraction and purification process of response surface optimization high-purity Caulis et Folium Chrysanthemi segeti total flavones is provided.
To achieve these goals, the technical scheme taked of the present invention is as follows:
A method for extraction and purification for response surface optimization high-purity Caulis et Folium Chrysanthemi segeti total flavones, its key technology point is, comprises the steps:
Step one: slightly carrying of Caulis et Folium Chrysanthemi segeti total flavones
Choose Caulis et Folium Chrysanthemi segeti, pulverize, take Caulis et Folium Chrysanthemi segeti powder, by 1:30-35 (g:mL) solid-liquid ratio, add the alcoholic solution that extractant volume fraction is 60%-70%, at 20-30 DEG C, to mixed liquor ultrasonic Treatment 50-70min under 80khz 200W condition, be placed in 60 DEG C of thermostat water bath lixiviate 0.5-1.5h, centrifugal, get supernatant, obtain crude extract;
Step 2: aqueous two-phase extraction
Crude extract step one obtained, adds C
2h
5oH-(NH
4)
2sO
4the double-aqueous phase system formed, described C
2h
5the mass fraction of OH is 20%-28%, described (NH
4)
2sO
4mass fraction be 16%-24%, add the NaCl that mass fraction is 1.5%-3%, add the HP-β-CD that mass fraction is 0.3-0.5%, adjust ph is 6-8, mixing, phase-splitting;
Take out be rich in Caulis et Folium Chrysanthemi segeti total flavones ethanol on phase, carry out distilling under reduced pressure, concentrated solution, obtain the Caulis et Folium Chrysanthemi segeti flavone ethanol extract of 0.25-2.00mg/ml different quality concentration;
Step 3: the purification of Caulis et Folium Chrysanthemi segeti total flavones
(1) macroporous adsorbent resin pretreatment
(2) macroporous adsorbent resin separation and purification
The Caulis et Folium Chrysanthemi segeti flavone ethanol extract of the 0.25-2.00mg/ml different quality concentration that step 2 is obtained, the different pH value of 4-9 are adjusted to acid or alkali, join in D-101 macroporous resin column, eluting is carried out with the different flow velocity of 1. 0-5.0mL/min with the eluant ethanol of the different volume fraction of 55%-95%, eluent is concentrated, obtains extracting solution;
(3) Their Determination by Spectrophotometry general flavone content is used
The absorption extracting solution 1mL that step (2) obtains, mend to 5mL with 70% ethanol, adding concentration is 5%NaN0
20.3mL, shakes up and puts 6min; Adding concentration is again 10%A1 (NO
3)
30.3mL, shakes up and puts 6min; Adding concentration is 4%NaOH4mL, is settled to l0mL with distilled water, measures absorbance after shaking up 15min, the regression equation according to absorbance (Y) and concentration (X): y=9.12x-0.023, R
2=0.9986, calculating concentration;
(4) experimental design and statistical analysis
A single factor experiment
Change sample concentration successively, loading pH, eluant strength, flow velocity carry out single factor experiment, by the concentration of the total flavones in the Caulis et Folium Chrysanthemi segeti total flavones extracting solution of Their Determination by Spectrophotometry gained in step (3);
B response phase method optimal design
According to single factor experiment result, select sample concentration, loading pH, eluant strength, flow velocity four factors, Design-Expert software is utilized to carry out experimental design according to Box-Benhnken design principle, with sample concentration A, loading pH B, eluant strength C, elution flow rate D for independent variable, with the TFCL response rate for response value Y, set up polynary quadratic equation:
Y=63.31-0.72A-0.83B-0.14C-0.072D-0.71AB-0.053AC-0.7AD-0.41BC+0.54BD-0.21CD-2.500E-0.78A
2-0.53B
2-2.22C
2-0.71D
2
(5) interpretation and optimization
Utilize Design-Expert software to carry out plot analysis according to polynary quadratic regression equation, obtain response surface and the contour map thereof of regression equation.
Further improve as the present invention, the macroporous adsorbent resin preprocess method of described step (1) is specific as follows:
Soak 24h completely with 95% ethanol macroporous adsorbent resin, adopt wet method dress post, with 95% ethanol cyclic washing, until water and effluent do not produce white opacity, then be washed till without alcohol taste with distilled water; Then successively with the hydrochloric acid of volume fraction 5% and the Strong oxdiative sodium drip washing repeatedly of mass fraction 2%, and static put 2-4h after, be washed till pH value neutrality with distilled water, be soaked in distilled water for subsequent use.
Further improve as the present invention, described step (2) macroporous adsorbent resin separation and purification is specific as follows:
Mass concentration is respectively 0.25,0.50,0.75,1.00,1.25,1.50 and the Caulis et Folium Chrysanthemi segeti flavone ethanol extract that obtains of the step 2 of 2.00mg/ml, be adjusted to pH value with acid or alkali and be respectively 4,5,6,7,8,9, join in D-101 macroporous resin column, the eluant ethanol being respectively 55%, 65%, 75%, 85%, 95% by volume fraction with 1.0,2.0,3.0,4.0, the flow velocity of 5.0mL/min carries out eluting, eluent is concentrated.
" NaCl that mass fraction is 1.5%-3% is added; add the HP-β-CD that mass fraction is 0.3-0.5% " in the present invention, refer to that " mass fraction of NaCl in aqueous two-phase is 1.5%-3%, and the mass fraction of HP-β-CD in aqueous two-phase is 0.3-0.5%.”
In order to obtain highly purified Caulis et Folium Chrysanthemi segeti total flavones, inventors performed large quantifier elimination.In plant known in those skilled in the art, the extracting method of total flavones is more, such as ultrasonic assistant extraction method, soxhlet extraction, water bath reflux method, ultrasonic synergistic combined-enzyme method, microwave loss mechanisms etc.Inventor adopts conventional extracting method to extract Caulis et Folium Chrysanthemi segeti total flavones, although can obtain the Caulis et Folium Chrysanthemi segeti total flavones liquid of certain content, extracts result and makes inventor dejected.Because the Various Complex composition of Caulis et Folium Chrysanthemi segeti itself affected, the purity of the Caulis et Folium Chrysanthemi segeti total flavones extracting solution adopting conventional extracting flavonoids technique to obtain is not high.In order to expect highly purified Caulis et Folium Chrysanthemi segeti total flavones, inventor enters new one and takes turns thinking.Inventor has to carry out separation and purification to the Caulis et Folium Chrysanthemi segeti total flavones extracting solution obtained, macroporous resin separation and purification is method conventional in compound extraction purification, simply crude extract can be carried out separation and purification, inventor is joyful in above-mentioned design, but after putting into practice, practical situation makes inventor disappointed.Inventor attempts having used multiple macroporous resin: D-101, AB-8, DA-201, SD-401 and DM-301, in any case adjustment sample concentration, pH, eluant kind and concentration and velocity separation purification result all undesirable.When inventor wants to abandon, unexpected discovery, before with purification by macroporous resin, by the extract and separate effect adding HP-β-CD in double-aqueous phase system and can reach good, make extraction product that macroporous resin can be utilized to obtain purifies and separates, analyzing its reason is that impurity substances is optionally wrapped up by HP-β-CD, because the water solublity of hydroxypropyl beta cyclodextrin is large, impurity is trapped in lower mutually in, based on above-mentioned discovery, through rational experimental design, exploration many times and huge creative work have searched out a kind of method for extraction and purification of high-purity Caulis et Folium Chrysanthemi segeti total flavones, entirety defines technical scheme of the present invention.
Compared with prior art, the present invention obtains beneficial effect and is:
The present invention is directed to Caulis et Folium Chrysanthemi segeti itself, adopt ultrasonic assistant to extract, the technique of aqueous two-phase extraction, the extraction ratio of Caulis et Folium Chrysanthemi segeti total flavones is uprised, the extraction ratio step-down of other non-flavone components, and separation and purification can be carried out with macroporous resin, the Caulis et Folium Chrysanthemi segeti total flavones purity finally obtained is significantly improved.
The present invention utilizes the Responds Surface Methodology of Box-Benhnken Designs center combination design model, with four variables, 3 levels totally 29 testing sites (5 central points) just can draw optimum results, obtain best macroporous resin separation and purification process, the purity of Caulis et Folium Chrysanthemi segeti total flavones after raising purification.
Operating procedure of the present invention is simple, cost is low, easy to operate, the content of the final Caulis et Folium Chrysanthemi segeti total flavones obtained can bring up to more than 96.51%.
Accompanying drawing explanation
Accompanying drawing 1 is that loading pH, loading mass concentration are on the response surface graphics of the impact of the response rate;
Accompanying drawing 2 is that elution flow rate, loading mass concentration are on the response surface graphics of the impact of the response rate;
Accompanying drawing 3 is that elution flow rate, loading pH are on the response surface graphics of the impact of the response rate;
Accompanying drawing 4 is that elution flow rate, loading mass concentration are on the response surface graphics of the impact of the response rate;
Accompanying drawing 5 is that elution flow rate, eluting concentration are on the response surface graphics of the impact of the response rate;
Accompanying drawing 6 is that eluting concentration, loading pH are on the response surface graphics of the impact of the response rate.
Detailed description of the invention
Below in conjunction with specific embodiment, detailed further describing is carried out to the present invention.
embodiment 1
A method for extraction and purification for response surface optimization high-purity Caulis et Folium Chrysanthemi segeti total flavones, its key technology point is, comprises the steps:
Step one: slightly carrying of Caulis et Folium Chrysanthemi segeti total flavones
Choose Caulis et Folium Chrysanthemi segeti, pulverize, take Caulis et Folium Chrysanthemi segeti powder 0.5Kg, add the alcoholic solution 15L that extractant volume fraction is 65%, at 25 DEG C, to mixed liquor ultrasonic Treatment 60min under 80khz 200W condition, be placed in 60 DEG C of thermostat water bath lixiviate 1h, centrifugal, get supernatant, obtain crude extract;
Step 2: aqueous two-phase extraction
Crude extract step one obtained, adds C
2h
5oH-(NH
4)
2sO
4the double-aqueous phase system formed, described C
2h
5the mass fraction of OH is 24%, described (NH
4)
2sO
4mass fraction be 18%, add the NaCl that mass fraction is 1.7%, add the HP-β-CD that mass fraction is 0.5%, adjust ph is 7, mixing, phase-splitting;
Take out be rich in Caulis et Folium Chrysanthemi segeti total flavones ethanol on phase, carry out distilling under reduced pressure, concentrated solution, obtain the Caulis et Folium Chrysanthemi segeti flavone ethanol extract of 0.25-2.00mg/ml different quality concentration;
Step 3: the purification of Caulis et Folium Chrysanthemi segeti total flavones
(1) macroporous adsorbent resin pretreatment
Get resin appropriate, 95% ethanol soaks 24h completely, removes macroporous resin fragment and foreign material.Adopt wet method dress post, with 95% ethanol cyclic washing, until water and effluent do not produce white opacity, then be washed till without alcohol taste with distilled water; Then successively with the hydrochloric acid of volume fraction 5% and the Strong oxdiative sodium drip washing repeatedly of mass fraction 2%, and static put 2-4h after, be washed till pH value neutrality with distilled water, be soaked in distilled water for subsequent use.
(2) macroporous adsorbent resin separation and purification
Mass concentration is respectively 0.25,0.50,0.75,1.00,1.25,1.50 and the Caulis et Folium Chrysanthemi segeti flavone ethanol extract that obtains of 2.00mg/ml step 2, be adjusted to pH value with acid or alkali and be respectively 4,5,6,7,8,9, join in D-101 macroporous resin column, loading volume is 60ml, the eluant ethanol being respectively 55%, 65%, 75%, 85%, 95% by volume fraction with 1.0,2.0,3.0,4.0, the flow velocity of 5.0mL/min carries out eluting, eluent is concentrated, obtains extracting solution;
(3) Their Determination by Spectrophotometry general flavone content is used
The absorption extracting solution 1mL that step (2) obtains, mend to 5mL with 70% ethanol, adding concentration is 5%NaN0
2aqueous solution 0.3mL, shake up and put 6min; Adding concentration is again 10%A1 (NO
3)
3aqueous solution 0.3mL, shake up and put 6min; Add the aqueous solution 4mL that concentration is 4%NaOH, be settled to l0mL with distilled water, after shaking up 15min, measure absorbance, the regression equation according to absorbance (Y) and concentration (X): y=9.12x-0.023, R
2=0.9986, calculating concentration; Aqueous solution
(4) experimental design and statistical analysis
A single factor experiment
Change sample concentration successively, loading pH, eluant strength, flow velocity carry out single factor experiment, with the content of the total flavones in the Caulis et Folium Chrysanthemi segeti total flavones extracting solution of Their Determination by Spectrophotometry gained in step (3);
B response phase method optimal design
According to single factor experiment result, to sample concentration, loading pH, eluant strength, flow velocity four factors, Design-Expert software is utilized to carry out experimental design according to Box-Benhnken design principle, with sample concentration A, loading pH B, eluant strength C, elution flow rate D is independent variable, with the TFCL response rate for response value Y, response surface experiments plan and test the results are shown in Table 1, response surface, curved surface variance analysis was in table 2, through Regression Model Simulator, the available following polynary quadratic equation of impact of three factor pair response value Y: Y=63.31-0.72A-0.83B-0.14C-0.072D-0.71AB-0.053AC-0.7AD-0. 41BC+
0.54BD-0.21CD-2.500E-0.78A
2-0.53B
2-2.22C
2-0.71D
2。
Analyzed from table 1, (
p=0.047<
p=0.05) quadratic regression equation model is remarkable, lose intend item not significantly (
p=0.428>
p=0.05).Known by the variance analysis of this model: loading pH(B) on the TFCL response rate affect significant difference (
p=0.047), and sample concentration (A), eluting concentration (C) and elution speed (D) three factors to affect difference to the TFCL response rate not remarkable.That is: the change of sample solution pH will the response rate of appreciable impact TFCL.Four factors are to the size pH(B successively of TFCL response rate influential effect) > sample concentration (A) > eluting concentration (C) > elution speed (D).
(5) interpretation and optimization
Utilize Design-Expert software to carry out plot analysis according to polynary quadratic regression equation, obtain response surface and the contour map thereof of regression equation.
As shown in figures 1 to 6, the response surface of the response rate, Open Side Down, and each and every one restraining factors of response rate Y and four present obvious second-degree parabola relation.Along with the increase of each factor, response value response rate Y is also in increase.According to lixiviate kinetic theory, along with pH(B), the increase of sample concentration (A), eluting concentration (C), elution speed (D), there is maximum in response value response rate Y, after increase again along with A, B, C, D, extraction yield Y presents the decline of Different Slope, this regression model has point of safes, and point of safes is maximum.By even experiment design in Design-Expert Software 8.0, TFF extraction yield is estimated, extreme value analysis is carried out to second-degree parabola function model, predict the best of breed of four factors: sample concentration 0.86mg/ml, pH=6, eluting concentration 74.27% and elution flow rate 2.23ml/min now model prediction maximum Y=87.3667%(P=0.994).
Through under above-mentioned optimum combination condition, carry out three groups of repeated trials, the content recording the Caulis et Folium Chrysanthemi segeti total flavones that the present embodiment method obtains is 96.51%.
comparative example 1
A method for extraction and purification for response surface optimization high-purity Caulis et Folium Chrysanthemi segeti total flavones, comprises the steps:
Step one: slightly carrying of Caulis et Folium Chrysanthemi segeti total flavones
Choose Caulis et Folium Chrysanthemi segeti, pulverize, take Caulis et Folium Chrysanthemi segeti powder 0.5Kg, add the alcoholic solution 15L that extractant volume fraction is 65%, at 25 DEG C, to mixed liquor ultrasonic Treatment 60min under 80khz 200W condition, be placed in 60 DEG C of thermostat water bath lixiviate 1h, centrifugal, get supernatant, obtain crude extract, make the Caulis et Folium Chrysanthemi segeti flavone ethanol extract of 0.86mg/ml.
Step 2: the purification of Caulis et Folium Chrysanthemi segeti total flavones
(1) macroporous adsorbent resin pretreatment
Get resin appropriate, 95% ethanol soaks 24h completely, removes macroporous resin fragment and foreign material.Adopt wet method dress post, with 95% ethanol cyclic washing, until water and effluent do not produce white opacity, then be washed till without alcohol taste with distilled water; Then successively with the hydrochloric acid of volume fraction 5% and the Strong oxdiative sodium drip washing repeatedly of mass fraction 2%, and static put 2-4h after, be washed till pH value neutrality with distilled water, be soaked in distilled water for subsequent use.
(2) macroporous adsorbent resin separation and purification
The Caulis et Folium Chrysanthemi segeti flavone ethanol extract that step one is obtained, be adjusted to pH value and be respectively 6, join in D-101 macroporous resin column, loading volume is 60ml, the eluant ethanol being respectively 74.29% by volume fraction carries out eluting with the flow velocity of 2.23mL/min, concentrate eluent, recording Caulis et Folium Chrysanthemi segeti general flavone content is 54.32%.
comparative example 2
A method for extraction and purification for response surface optimization high-purity Caulis et Folium Chrysanthemi segeti total flavones, comprises the steps:
Step one: slightly carrying of Caulis et Folium Chrysanthemi segeti total flavones
Choose Caulis et Folium Chrysanthemi segeti, pulverize, take Caulis et Folium Chrysanthemi segeti powder 0.5Kg, add the alcoholic solution 15L that extractant volume fraction is 65%, at 25 DEG C, to mixed liquor ultrasonic Treatment 60min under 80khz 200W condition, be placed in 60 DEG C of thermostat water bath lixiviate 1h, centrifugal, get supernatant, obtain crude extract;
Step 2: aqueous two-phase extraction
Crude extract step one obtained, adds C
2h
5oH-(NH
4)
2sO
4the double-aqueous phase system formed, described C
2h
5the mass fraction of OH is 24%, described (NH
4)
2sO
4mass fraction be 18%, add the NaCl that mass fraction is 1.7%, add the HP-β-CD that mass fraction is 0.5%, adjust ph is 7, mixing, phase-splitting;
Take out be rich in Caulis et Folium Chrysanthemi segeti total flavones ethanol on phase, carry out distilling under reduced pressure, concentrated solution, obtain the Caulis et Folium Chrysanthemi segeti flavone ethanol extract of 0.86mg/ml different quality concentration;
Step 3: the purification of Caulis et Folium Chrysanthemi segeti total flavones
(1) macroporous adsorbent resin pretreatment
Get resin appropriate, 95% ethanol soaks 24h completely, removes macroporous resin fragment and foreign material.Adopt wet method dress post, with 95% ethanol cyclic washing, until water and effluent do not produce white opacity, then be washed till without alcohol taste with distilled water; Then successively with the hydrochloric acid of volume fraction 5% and the Strong oxdiative sodium drip washing repeatedly of mass fraction 2%, and static put 2-4h after, be washed till pH value neutrality with distilled water, be soaked in distilled water for subsequent use.
(2) macroporous adsorbent resin separation and purification
The Caulis et Folium Chrysanthemi segeti flavone ethanol extract that step 2 is obtained, be adjusted to pH value and be respectively 6, join in D-101 macroporous resin column, loading volume is 60ml, the eluant ethanol being respectively 74.29% by volume fraction carries out eluting with the flow velocity of 2.23mL/min, concentrate eluent, recording Caulis et Folium Chrysanthemi segeti general flavone content is 61.31%.
comparative example 3
A method for extraction and purification for response surface optimization high-purity Caulis et Folium Chrysanthemi segeti total flavones, comprises the steps:
Step one: slightly carrying of Caulis et Folium Chrysanthemi segeti total flavones
Choose Caulis et Folium Chrysanthemi segeti, pulverize, take Caulis et Folium Chrysanthemi segeti powder 0.5Kg, add the alcoholic solution 15L that extractant volume fraction is 65%, at 25 DEG C, to mixed liquor ultrasonic Treatment 60min under 80khz 200W condition, be placed in 60 DEG C of thermostat water bath lixiviate 1h, centrifugal, get supernatant, obtain crude extract;
Step 2: aqueous two-phase extraction
Crude extract step one obtained, adds C
2h
5oH-(NH
4)
2sO
4the double-aqueous phase system formed, described C
2h
5the mass fraction of OH is 24%, described (NH
4)
2sO
4mass fraction be 18%, add the NaCl that mass fraction is 1.7%, add the HP-β-CD that mass fraction is 0.5%, adjust ph is 7, mixing, phase-splitting;
Take out be rich in Caulis et Folium Chrysanthemi segeti total flavones ethanol on phase, carry out distilling under reduced pressure, concentrated solution, the content measuring Caulis et Folium Chrysanthemi segeti total flavones is 62.82%.
The above embodiment is only the preferred embodiments of the present invention, and and the feasible enforcement of non-invention exhaustive.For persons skilled in the art, to any apparent change done by it under the prerequisite not deviating from the principle of the invention and spirit, all should be contemplated as falling with within claims of the present invention.
Claims (3)
1. a method for extraction and purification for response surface optimization high-purity Caulis et Folium Chrysanthemi segeti total flavones, is characterized in that, comprise the steps:
Step one: slightly carrying of Caulis et Folium Chrysanthemi segeti total flavones
Choose Caulis et Folium Chrysanthemi segeti, pulverize, take Caulis et Folium Chrysanthemi segeti powder, by 1:30-35 (g:mL) solid-liquid ratio, add the alcoholic solution that extractant volume fraction is 60%-70%, at 20-30 DEG C, to mixed liquor ultrasonic Treatment 50-70min under 80khz 200W condition, be placed in 60 DEG C of thermostat water bath lixiviate 0.5-1.5h, centrifugal, get supernatant, obtain crude extract;
Step 2: aqueous two-phase extraction
Crude extract step one obtained, adds C
2h
5oH-(NH
4)
2sO
4the double-aqueous phase system formed, described C
2h
5the mass fraction of OH is 20%-28%, described (NH
4)
2sO
4mass fraction be 16%-24%, add the NaCl that mass fraction is 1.5%-3%, add the HP-β-CD that mass fraction is 0.3-0.5%, adjust ph is 6-8, mixing, phase-splitting;
Take out be rich in Caulis et Folium Chrysanthemi segeti total flavones ethanol on phase, carry out distilling under reduced pressure, concentrated solution, obtain the Caulis et Folium Chrysanthemi segeti flavone ethanol extract of 0.25-2.00mg/ml different quality concentration;
Step 3: the purification of Caulis et Folium Chrysanthemi segeti total flavones
(1) macroporous adsorbent resin pretreatment
(2) macroporous adsorbent resin separation and purification
The Caulis et Folium Chrysanthemi segeti flavone ethanol extract of the 0.25-2.00mg/ml different quality concentration that step 2 is obtained, the different pH value of 4-9 are adjusted to acid or alkali, join in D-101 macroporous resin column, eluting is carried out with the different flow velocity of 1. 0-5.0mL/min with the eluant ethanol of the different volume fraction of 55%-95%, eluent is concentrated, obtains extracting solution;
(3) Their Determination by Spectrophotometry general flavone content is used
The absorption extracting solution 1mL that step (2) obtains, mend to 5mL with 70% ethanol, adding concentration is 5%NaN0
20.3mL, shakes up and puts 6min; Adding concentration is again 10%A1 (NO
3)
30.3mL, shakes up and puts 6min; Adding concentration is 4%NaOH4mL, is settled to l0mL with distilled water, measures absorbance after shaking up 15min, the regression equation according to absorbance (Y) and concentration (X): y=9.12x-0.023, R
2=0.9986, calculating concentration;
(4) experimental design and statistical analysis
A single factor experiment
Change sample concentration successively, loading pH, eluant strength, flow velocity carry out single factor experiment, by the concentration of the total flavones in the Caulis et Folium Chrysanthemi segeti total flavones extracting solution of Their Determination by Spectrophotometry gained in step (3);
B response phase method optimal design
According to single factor experiment result, select sample concentration, loading pH, eluant strength, flow velocity four factors, Design-Expert software is utilized to carry out experimental design according to Box-Benhnken design principle, with sample concentration A, loading pH B, eluant strength C, elution flow rate D for independent variable, with the TFCL response rate for response value Y, set up polynary quadratic equation:
Y=63.31-0.72A-0.83B-0.14C-0.072D-0.71AB-0.053AC-0.7AD-0.41BC+0.54BD-0.21CD-2.500E-0.78A
2-0.53B
2-2.22C
2-0.71D
2
(5) interpretation and optimization
Utilize Design-Expert software to carry out plot analysis according to polynary quadratic regression equation, obtain response surface and the contour map thereof of regression equation.
2. the method for extraction and purification of a kind of response surface optimization high-purity Caulis et Folium Chrysanthemi segeti total flavones according to claim 1, it is characterized in that, the macroporous adsorbent resin preprocess method of described step (1) is specific as follows:
Soak 24h completely with 95% ethanol macroporous adsorbent resin, adopt wet method dress post, with 95% ethanol cyclic washing, until water and effluent do not produce white opacity, then be washed till without alcohol taste with distilled water; Then successively with the hydrochloric acid of volume fraction 5% and the Strong oxdiative sodium drip washing repeatedly of mass fraction 2%, and static put 2-4h after, be washed till pH value neutrality with distilled water, be soaked in distilled water for subsequent use.
3. the method for extraction and purification of a kind of response surface optimization high-purity Caulis et Folium Chrysanthemi segeti total flavones according to claim 1 and 2, it is characterized in that, described step (2) macroporous adsorbent resin separation and purification is specific as follows:
Mass concentration is respectively 0.25,0.50,0.75,1.00,1.25,1.50 and the Caulis et Folium Chrysanthemi segeti flavone ethanol extract that arrives of the step 2 of 2.00mg/ml, be adjusted to pH value with acid or alkali and be respectively 4,5,6,7,8,9, join in D-101 macroporous resin column, the eluant ethanol being respectively 55%, 65%, 75%, 85%, 95% by volume fraction with 1.0,2.0,3.0,4.0, the flow velocity of 5.0mL/min carries out eluting, eluent is concentrated.
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