CN105030869A - Bo-chrysanthemum total flavone extract preparation method based on response surface optimization - Google Patents
Bo-chrysanthemum total flavone extract preparation method based on response surface optimization Download PDFInfo
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Abstract
The invention discloses a Bo-chrysanthemum total flavone extract preparation method based on response surface optimization. The preparation method is characterized by comprises the following steps: grinding Bo-chrysanthemum, adding ethanol, carrying out reflux extraction, cooling the extract, subjecting the extract to centrifugation, and collecting the supernate namely the Bo-chrysanthemum total flavone extract; wherein the optimal conditions that influence the reflux extraction effect are determined through response surface experiments. The provided extraction method has the advantages that the operation is convenient and fast, the extraction cost is low, and no toxic or harmful substance is introduced into the final product (Bo-chrysanthemum wine). The optimal extraction factors are determined by the response surface design and optimization, and thus the optimal extraction factors are scientific.
Description
Technical field
The present invention relates to the extracting method of total flavones in a kind of Flos Chrysanthemi, be specifically related to a kind of extracting method based on total flavones in the Flos Chrysanthemi of response surface optimization.
Background technology
Health-care white spirit has the history of thousands of years, is the important component part of scientific tcm.Along with the progress of science and technology, from the basis of the traditional handicraft of Traditional Chinese medicinal wine, develop into modernization meanses such as utilizing extraction, lixiviate and biological engineering, extract the functional health health promoting wine that the effective ingredient in Chinese medicine is made.
As the Flos Chrysanthemi of one of the typical authentic medicinal herbs in Bozhou and four your name chrysanthemums, the mainly Flavonoid substances of effective ingredient wherein, has wind-dispelling heat-dissipating, expelling summer-heat improving eyesight, coronary artery dilator, reduces blood pressure, prevents hyperlipidemia, antibacterial, antiviral, antiinflammatory, anti-ageing effect of waiting for a long time.The functional components of Flos Chrysanthemi and wine are merged effectively, obtains the Flos Chrysanthemi health promoting wine with best nutritional formula and comfortable mouthfeel significant, and the functional components in Flos Chrysanthemi is extracted as much as possible, particularly important.Flavone compound, owing to having medical active widely, is often used to the important indicator evaluating Flos Chrysanthemi extract quality.
The Flos Chrysanthemi extracting process of current bibliographical information has decocting method, soda acid extraction, ultrasonic extraction and supercritical extraction.The extraction ratio of decocting method is lower, the novel extraction process such as ultrasonic extraction and supercritical extraction is subject to extensive concern because extraction efficiency is higher, but excessive owing to there is investment aborning, problems such as complex operation and limit said method popularization and application in the industrial production.And alcohol reflux, there is noiselessness, pollution-free, energy consumption is low, extraction ratio high, belong to eco-friendly extracting method.
Summary of the invention
The present invention is for overcoming the deficiencies in the prior art, and providing a kind of take ethanol as the preparation method of the Flos Chrysanthemi total flavones extracting solution of Extraction solvent.
The present invention applies response surface design software and carries out response surface condition optimizing to concentration of alcohol, extraction time, extraction time three factors, obtains preferably extracting method.
Technical solution problem of the present invention, adopts following technical scheme:
The present invention is based on the preparation method of the Flos Chrysanthemi total flavones extracting solution of response surface optimization, its feature is to carry out as follows:
(1) Flos Chrysanthemi is ground into Flos Chrysanthemi coarse powder by pulverizer;
(2) ethanol adding 50% ~ 90% in Flos Chrysanthemi coarse powder extracts 1 ~ 3 time with the temperature reflux of 50 DEG C ~ 90 DEG C, extracts 1 ~ 3.5h, merge extractive liquid, at every turn; The ratio of described Flos Chrysanthemi meal quality and reflux, extract, total ethanol volume used is 1:8 ~ 20g/mL;
(3) by extracting solution cool, centrifugal, gained supernatant is Flos Chrysanthemi total flavones extracting solution.
Preferably, the preparation method that the present invention is based on the Flos Chrysanthemi total flavones extracting solution of response surface optimization is carried out as follows:
(1) Flos Chrysanthemi is ground into Flos Chrysanthemi coarse powder by pulverizer;
(2) ethanol adding 78% in Flos Chrysanthemi coarse powder extracts 2 times with the temperature reflux of 85 DEG C, merge extractive liquid; During first time reflux, extract, Flos Chrysanthemi meal quality is 1:7g/mL with the ratio of ethanol contend used, and extraction time is 2h; During second time reflux, extract, Flos Chrysanthemi meal quality is 1:3g/mL with the ratio of ethanol contend used, and extraction time is 1.5h;
(3) by extracting solution cool, centrifugal, gained supernatant is Flos Chrysanthemi extractive of general flavone.
Wherein, step (3) described centrifugal be at 15 DEG C, with the centrifugation 10min of 8000r/min.
By optimization method of the present invention, in the Flos Chrysanthemi total flavones extracting solution finally obtained, the content of total flavones is 0.1277g/L.
Compared with prior art, good effect of the present invention is:
Method of the present invention is simple to operate, and solvent for use is ethanol, non-toxic inexpensive, and in gained Flos Chrysanthemi total flavones extracting solution, general flavone content reaches 0.1277g/L.
The present invention is optimized the extraction process of total flavones in Flos Chrysanthemi by Responds Surface Methodology: first utilize that single factor design investigates concentration of alcohol, (Flos Chrysanthemi meal quality (unit be g) with the ratio of ethanol contend used (unit is mL)), Extracting temperature, extraction time, extraction time five factors are on the impact of extracting flavonoids amount for solid-liquid ratio.On the impact of extracting flavonoids amount significantly, recycling response surface experimental design investigates above-mentioned 3 factors, obtains the optimum extraction condition of total flavones in Flos Chrysanthemi for comprehensive discovery concentration of alcohol, extraction time, extraction time.
Accompanying drawing explanation
Fig. 1 is the variance analysis of extracting data in embodiment 2;
Fig. 2 is the residual error Normal P-P extracting data in embodiment 2;
Fig. 3 is predictive value and actual value corresponding relation figure in embodiment 2;
Fig. 4 is the contour plots analysis of each factor in embodiment 2, by contour plots analysis that is left-to-right, that be respectively AB factor, BC factor, AC factor and C factor from top to bottom;
Fig. 5 is the optimum organization that in embodiment 2, response surface extraction is optimized.
Detailed description of the invention
The detailed description of the invention of the preparation method of the Flos Chrysanthemi total flavones extracting solution that the present invention is based on response surface optimization is below provided.
Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.In addition should be understood that those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the application's appended claims limited range equally after the content of having read the present invention's instruction.
In following embodiment, in gained Flos Chrysanthemi extractive of general flavone, Determination Method of Flavone Content is as follows:
(1) instrument and reagent
Shanghai Yuan Xi Instrument Ltd. UV-9000 spectrophotometer; Control substance of Rutin; Water is ultra-pure water, and other reagent is analytical pure.
(2) testing conditions
Determined wavelength: 510nm.
(3) preparation of reference substance solution
Precision take be dried to constant weight anhydrous rutin 10mg in 50mL volumetric flask, add appropriate 95% dissolve with ethanol and standardize solution, shake up, obtain 0.200mg/mL rutin contrast mother solution.
(4) foundation of standard curve
Precision measures rutin contrast mother solution 2.0mL, 4.0mL, 6.0mL, 8.0mL, 10.0mL, 12.0mL, 14.0mL, 16.0mL are placed in 25mL volumetric flask respectively, add 5% sodium nitrite solution 1mL, mixing, places 6min, adds 10% aluminum nitrate solution 1mL, shake up, place 6min, add 10% sodium hydroxide solution 10mL, be settled to scale with 30% ethanol, shake up, leave standstill.After 15min, the absorbance under test 510nm.Reference is made with the blank solution not containing rutin.Take absorbance as vertical coordinate, control substance of Rutin concentration is abscissa, drawing standard curve, and calculates regression equation and be: Y=0.0850X-0.0006.Correlation coefficient is 0.9972.
(5) assay
Draw extracting solution 10mL, dissolve with concentration of alcohol when extracting and be settled to 50mL, shaking up, obtain liquid to be measured.Draw 3mL liquid to be measured in 25mL volumetric flask, add 5% sodium nitrite solution 1mL, mixing, place 6min, add 10% aluminum nitrate solution 1mL, shake up, place 6min, add 10% sodium hydroxide solution 10mL, be settled to scale with 30% ethanol, shake up, leave standstill.After 15min, the absorbance under test 510nm.Reference is made with the blank solution not containing extracting solution.Combined standard curve, namely obtains the content of total flavones in extracting solution.
Embodiment 1
The present embodiment, according to method provided by the invention, selects different condition to carry out the extraction optimization of total flavones in Flos Chrysanthemi.
1, concentration of alcohol is on the impact of general flavone content in Flos Chrysanthemi total flavones extracting solution
Take 10g Flos Chrysanthemi coarse powder, be respectively 50% by 100mL concentration, 60%, 70%, 80%, the alcoholic solution of 90% is as extraction solvent, reflux, extract, 2h at 70 DEG C, after the cooling of gained extracting solution at 15 DEG C, with the centrifugation 10min of 8000r/min, obtains corresponding Flos Chrysanthemi total flavones extracting solution.Measure absorbance and the general flavone content of gained extracting solution as stated above, as shown in table 1.Can find out, when concentration of alcohol is 80%, in extracting solution, the content of total flavones is maximum.
The absorbance of gained Flos Chrysanthemi total flavones extracting solution and general flavone content under table 1 different ethanol concentration
| Concentration of alcohol | Absorbance | General flavone content (g/L) |
| 50％ | 0.9610 | 0.0811 |
| 60％ | 1.0496 | 0.0886 |
| 70％ | 1.1455 | 0.0968 |
| 80％ | 1.2355 | 0.1044 |
| 90％ | 0.8654 | 0.0730 |
2, solid-liquid ratio is on the impact of general flavone content in Flos Chrysanthemi total flavones extracting solution
Take 10g Flos Chrysanthemi coarse powder, use 80mL respectively, 100mL, 120mL, 140mL, 160mL, 180mL, 200mL (namely solid-liquid ratio is respectively 1:8,1:10,1:12,1:14,1:16,1:18,1:20) concentration be the alcoholic solution of 80% as extraction solvent, reflux, extract, 2h at 70 DEG C, after the cooling of gained extracting solution at 15 DEG C, with the centrifugation 10min of 8000r/min, obtain corresponding Flos Chrysanthemi total flavones extracting solution.Measure absorbance and the general flavone content of gained extracting solution as stated above, as shown in table 2.
The interaction effect of solid-liquid ratio is not obvious as can be seen from the table.Along with the increase of solid-liquid ratio, the dissolution rate of other materials such as Flos Chrysanthemi Determination of Chlorophyll is accelerated, and extracting solution color is partially green.Total flavones dissolution rate slows down relatively, therefore the content of total flavones is on a declining curve.The immersion degree of comprehensive Flos Chrysanthemi and consume the amount cost of liquid, determine that solid-liquid ratio is 1:10.
The absorbance of gained Flos Chrysanthemi total flavones extracting solution and general flavone content under the different ethanol consumption of table 2
| Ethanol consumption (mL) | Absorbance | General flavone content (g/L) |
| 80 | 1.4391 | 0.1217 |
| 100 | 1.3029 | 0.1101 |
| 120 | 1.1186 | 0.0945 |
| 140 | 1.0275 | 0.0867 |
| 160 | 0.9819 | 0.0829 |
| 180 | 0.8916 | 0.0752 |
| 200 | 0.7073 | 0.0595 |
3, Extracting temperature is on the impact of general flavone content in Flos Chrysanthemi total flavones extracting solution
Take 10g Flos Chrysanthemi coarse powder, be that the alcoholic solution of 80% is as Extraction solvent by 100mL concentration, 5 thermogrades are set, are respectively 50 DEG C, 60 DEG C, 70 DEG C, 80 DEG C, 90 DEG C, reflux, extract, 2h at the temperature disclosed above, after the cooling of gained extracting solution at 15 DEG C, with the centrifugation 10min of 8000r/min, obtain corresponding Flos Chrysanthemi total flavones extracting solution.Measure absorbance and the general flavone content of gained extracting solution as stated above, as shown in table 3.
The absorbance of gained Flos Chrysanthemi total flavones extracting solution and general flavone content under the different Extracting temperature of table 3
| Extracting temperature | Absorbance | General flavone content (g/L) |
| 50℃ | 0.4455 | 0.0373 |
| 60℃ | 0.6052 | 0.0508 |
| 70℃ | 0.8037 | 0.0677 |
| 80℃ | 0.9885 | 0.0834 |
| 90℃ | 1.3550 | 0.1146 |
4, extraction time is on the impact of general flavone content in Flos Chrysanthemi total flavones extracting solution
Take 10g Flos Chrysanthemi coarse powder, with 100mL concentration be the alcoholic solution of 80% as Extraction solvent, respectively lixiviate 1h, 1.5h at 70 DEG C, 2h, 2.5h, 3h, 3.5h, after the cooling of gained extracting solution at 15 DEG C, with the centrifugation 10min of 8000r/min, obtains corresponding Flos Chrysanthemi total flavones extracting solution.Measure absorbance and the general flavone content of gained extracting solution as stated above, as shown in table 4.
The absorbance of gained Flos Chrysanthemi total flavones extracting solution and general flavone content under table 4 different extraction time
| Extraction time | Absorbance | General flavone content (g/L) |
| 1h | 1.0433 | 0.0881 |
| 1.5h | 1.1420 | 0.0965 |
| 2h | 1.1940 | 0.1009 |
| 2.5h | 1.2222 | 0.1033 |
| 3h | 1.3599 | 0.1150 |
| 3.5h | 1.3850 | 0.1171 |
5, extraction time is on the impact of general flavone content in Flos Chrysanthemi extracting solution
Take 10g Flos Chrysanthemi coarse powder, be 100mL concentration by total amount be that the alcoholic solution of 80% is as Extraction solvent, the solid-liquid ratio set by table 5 and extraction time different number of times of lixiviate at 70 DEG C, after the cooling of gained extracting solution at 15 DEG C, with the centrifugation 10min of 8000r/min, obtain corresponding Flos Chrysanthemi total flavones extracting solution.Measure absorbance and the general flavone content of gained extracting solution as stated above, as shown in table 6.Can find out that extraction twice rear absorbance tends towards stability, slightly rise, to extract 2 times for optimum extraction number of times.
Solid-liquid ratio and the setting of extraction time under the different extraction time of table 5
| Extracting times | Once | Secondary | Three times | Four times |
| 1 | 10 times amount lixiviate 1.5h | 7 times amount lixiviate 1h | 6 times amount lixiviate 1h | 5 times amount lixiviate 0.5h |
| 2 | 3 times amount lixiviate 0.5h | 2 times amount lixiviate 0.3h | 2 times amount lixiviate 0.5h | |
| 3 | 2 times amount lixiviate 0.2h | 2 times amount lixiviate 0.3h | ||
| 4 | 1 times amount lixiviate 0.2h |
The absorbance of gained Flos Chrysanthemi total flavones extracting solution and general flavone content under the different extraction time of table 6
| Extraction time | Absorbance | General flavone content (g/L) |
| 1 | 0.8848 | 0.0746 |
| 2 | 1.1024 | 0.0931 |
| 3 | 1.0961 | 0.0926 |
| 4 | 1.1436 | 0.0966 |
6, extraction time and ethanol times amount optimal proportion are selected
Take 10g Flos Chrysanthemi coarse powder, using 80% ethanol as Extraction solvent, the solid-liquid ratio (total amount of alcohol added is stabilized in 1:10g/mL, and namely solid-liquid ratio is 1:10) set by table 7, lixiviate twice at 70 DEG C, first time 2h, second time 1.5h.Lixiviating solution gauze is filtered dry for the first time, then pours remaining ethanol into.At 15 DEG C, with the centrifugation 10min of 8000r/min, obtain corresponding Flos Chrysanthemi total flavones extracting solution.Measure absorbance and the general flavone content of gained extracting solution as stated above, as shown in table 7.Add 7 times amount ethanol when can find out first time reflux, extract, when adding 3 times amount ethanol during second time reflux, extract, absorbance is maximum, and general flavone content is maximum.
The absorbance of gained Flos Chrysanthemi total flavones extracting solution and general flavone content under table 1 different ethanol concentration
| Ethanol times amount | Ethanol times amount | Ethanol times amount | Ethanol times amount | |
| For the first time | 5 | 6 | 7 | 8 |
| For the second time | 5 | 4 | 3 | 2 |
| Absorbance | 1.4585 | 1.3522 | 1.4882 | 1.3696 |
| General flavone content (g/L) | 0.1180 | 0.1089 | 0.1205 | 0.1104 |
Embodiment 2 response surface extracts design
In conjunction with the data that single factor test condition optimizing obtains, concentration of alcohol, extraction time, extraction time three factors are carried out to the response surface and extracted optimization.Arranging concentration of alcohol is 70%, 80%, 90% 3 level, and extraction time is 2.5h, 3h, 3.5h tri-levels, and extraction time is 1 time, 2 times, 3 times three levels, obtains 17 groups of data.
(1) variance analysis
Fig. 1 is the variance analysis schematic diagram of data, and probability (0.0020) <0.05 that model is corresponding, the equation significance of matching is good.Disappearance item (0.8109) >0.05, non-rationality index is not remarkable, and quadratic polynomial regression model is correct.Can these data be applicable to doing response surface optimization design.
(2) residual analysis
Fig. 2 is residual error Normal P-P, and figure mid point is better the closer to straight line.Fig. 3 is predictive value and actual value corresponding relation figure, and figure mid point is better the closer to straight line equally.
(3) contour plots analysis
Fig. 4 is the contour plots analysis of each factor, by contour plots analysis that is left-to-right, that be respectively AB factor, BC factor, AC factor and C factor from top to bottom.
Response surface extracts the checking optimized: optimize through response surface extraction the optimum organization obtained and see Fig. 5.
Therefore, optimized extraction method is:
(1) Flos Chrysanthemi is ground into Flos Chrysanthemi coarse powder by pulverizer;
(2) ethanol adding 78% in Flos Chrysanthemi coarse powder extracts 2 times with the temperature reflux of 85 DEG C, merge extractive liquid; During first time reflux, extract, Flos Chrysanthemi meal quality is 1:7g/mL with the ratio of ethanol contend used, and extraction time is 2h; During second time reflux, extract, Flos Chrysanthemi meal quality is 1:3g/mL with the ratio of ethanol contend used, and extraction time is 1.5h;
(3) gained extracting solution cooling after at 15 DEG C, with the centrifugation 10min of 8000r/min, obtain corresponding Flos Chrysanthemi total flavones extracting solution.
By best practice operation, do 3 Duplicate Samples, measure absorbance and be respectively 1.4123,1.3970,1.4112, design with the response surface absorbance obtained and differ in 10% scope, meet the requirements.
In sum, contrast according to the single factor test condition optimizing of concentration of alcohol, solid-liquid ratio, Extracting temperature, extraction time, extraction time five factors, response surface optimization is carried out to concentration of alcohol, extraction time, extraction time three factors, finally determine optimum organization, namely Extraction solvent made by the ethanol of 78%, extract at twice (7 times amount extract 2h, and 3 times amount extract 1.5h).
Extracting total flavones amount in the Flos Chrysanthemi total flavones extracting solution obtained according to best practice is 0.1277g/L.
Claims (3)
1., based on a preparation method for the Flos Chrysanthemi total flavones extracting solution of response surface optimization, it is characterized in that carrying out as follows:
(1) Flos Chrysanthemi is ground into Flos Chrysanthemi coarse powder by pulverizer;
(2) ethanol adding 50% ~ 90% in Flos Chrysanthemi coarse powder extracts 1 ~ 3 time with the temperature reflux of 50 DEG C ~ 90 DEG C, extracts 1 ~ 3.5h, merge extractive liquid, at every turn; The ratio of described Flos Chrysanthemi meal quality and reflux, extract, total ethanol volume used is 1:8 ~ 20g/mL;
(3) by extracting solution cool, centrifugal, gained supernatant is Flos Chrysanthemi total flavones extracting solution.
2. the preparation method of the Flos Chrysanthemi total flavones extracting solution based on response surface optimization according to claim 1, is characterized in that carrying out as follows:
(1) Flos Chrysanthemi is ground into Flos Chrysanthemi coarse powder by pulverizer;
(2) ethanol adding 78% in Flos Chrysanthemi coarse powder extracts 2 times with the temperature reflux of 85 DEG C, merge extractive liquid; During first time reflux, extract, Flos Chrysanthemi meal quality is 1:7g/mL with the ratio of ethanol contend used, and extraction time is 2h; During second time reflux, extract, Flos Chrysanthemi meal quality is 1:3g/mL with the ratio of ethanol contend used, and extraction time is 1.5h;
(3) by extracting solution cool, centrifugal, gained supernatant is Flos Chrysanthemi total flavones extracting solution.
3. the preparation method of the Flos Chrysanthemi total flavones extracting solution based on response surface optimization according to claim 1 and 2, is characterized in that: step (3) described centrifugal be at 15 DEG C, with the centrifugation 10min of 8000r/min.
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