CN103599147A - Wax myrtle leaf total-flavonoid extracting process and extracting condition optimization method thereof - Google Patents
Wax myrtle leaf total-flavonoid extracting process and extracting condition optimization method thereof Download PDFInfo
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Abstract
The invention discloses a wax myrtle leaf total-flavonoid extracting method which takes oxidation activity resistance as an index. The method comprises the steps of mixing ethanol and wax myrtle leaves according to the liquid/material ratio of (10-25)mL: 1g, extracting for 5-11 minutes at the temperature of 60-90 DEG C by adopting a microwave-assisted extraction method, carrying out centrifugation for 5 minutes at the rate of 3,500r/min, and extracting supernatant, thereby obtaining a wax myrtle leaf extract, wherein the extraction time is 7.00 minutes when the extraction temperature is 73.3 DEG C, the equivalent of Trolox in the extract assayed by using a DPPH (2,2-di-phenyl-1-picrylhydrazyl) method and an ABTS (2,2'-azinobis(3-ethylbenzothiazoline-6-sulphonic acid)-diammonium salt) method respectively is 1,352.10mu mol/g and 1,438.73mu mol/g when the liquid/material ratio is 20mL: 1g, and the content of total flavonoids of the extract is 70.94 mg/g. The invention further provides a method for optimizing a wax myrtle leaf total-flavonoid extracting process by using a response surface methodology, wherein oxidation activity resistance is taken as an index, three variables, namely extraction temperature, extraction time and liquid/material ratio, are set, and optimization is carried out by using Design Expert software according to Box-Behnken design principles. The method has the advantages that the cost is low, the extraction time is short, the extraction efficiency is high, and the source of raw materials is wide.
Description
Technical field
The invention belongs to medical technical field, relate to extraction process and optimization for extracting condition method thereof that a kind of Folium Myricae rubrae total flavones extracts, particularly relate to and take antioxidant activity as index, utilize Response Surface Method to optimize the method for Folium Myricae rubrae total flavones extraction conditions.
Background technology
Free radical, a kind of active group or molecule with non-matching electronics that human body produces while carrying out vital movement, is the intermediate product of normal biochemical process, at body, normally and in pathological process all exists.
Free radical in organism mainly comprises ultra-oxygen anion free radical (O
2-), hydroxyl radical free radical (OH) and lipid peroxidation free radical (ROO) etc., can attack the biomacromolecule in body, as nucleic acid, protein, polyunsaturated fatty acid, thereby body is produced and is poisoned.Free radicals injury is the key factor that causes Systemic aging and some disorders such as cancers, diabetes, cardiovascular disease etc.Therefore, the research and development of anti-oxidation active substance becomes the focus of Chinese scholars research.
Chemosynthesis antioxidant was once once being favored as dibenzylatiooluene (BHT), Butylated hydroxyanisole (BHA) etc., but find that by zoopery they have certain toxicity and teratogenesis, make people produce doubt to synthetized oxidation preventive agent.The Natural antioxidant is safe, nontoxic, meets people to healthy, safe new demand.Clinical researches show in a large number, and multiple natural plants comprises that vegetable, fruit, corn and Chinese herbal medicine etc. all contain the oxidation-resistant active ingredients such as flavone, polyphenol, tannin, saponin and alkaloid, have reduced the oxidative damage of body to a certain extent.
Folium Myricae rubrae is dicotyledonous guiding principle Myruca ceas
mycicacaemyrica(Myrica) Myrica L evergreen shrubs or dungarunga, formal name used at school
myrica rubra(Lour.) Sieb.et Zucc., English name China Waxmyrtle, the dry blade of China Bayberry.Record the herbal > > in < < China.Main chemical compositions is flavonoid and procyanidin class, and Flavonoid substances is ampelopsin, Quercetin, kaempferol and is connected with separately the glycosides that different sugar forms.The experiment of pharmacology and vivo and vitro confirms: Folium Myricae rubrae has good reduction blood glucose, antibacterial, anti-inflammatory and antalgic, hepatoprotective, antitumor and antioxidation.
Folium Myricae rubrae contains abundant Flavonoid substances, has very strong antioxidation.Current existing Folium Myricae rubrae total flavone extracting method is: use alcoholic solution lixiviate, in conjunction with ultrasonic householder method, at 45 times, after 40% heavy soak with ethanol 24 h of sample, ultrasonic auxiliary extraction 45 min, extract 2 times continuously; Ultrasonic-microwave synergistic extraction, extracts temperature 70 C, liquid ratio 25 mL/g, concentration of alcohol 60%, extraction times 40 min, extraction time 2 times; Extraction time is long, and extraction time is many.Response curve design method (Response Surface Methodology, RSM): be to utilize rational test design method and obtain by experiment a given data, adopt polynary quadratic regression equation to carry out the functional relationship between data fitting and response value, by the analysis to regression equation, seek optimal procedure parameters, solve a kind of statistical method (claiming again Fertilizer Test of Regression Design) of Multivariable.The present invention has done more deep research to the extraction of Folium Myricae rubrae and antioxidant activity, provides a kind of and saves time, efficient, the extracting method that solvent load is few.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind ofly take antioxidant activity as index, the Folium Myricae rubrae total flavone extracting process that uses response optimal method to obtain, the present invention is optimized extracting temperature, extraction time and these influence factors of liquid ratio, thereby improves the yield of total flavones.
In order to solve the problems of the technologies described above, the invention provides a kind of Folium Myricae rubrae total flavone extracting process that antioxidant activity is index of take, Folium Myricae rubrae mixes according to the liquid ratio of 10-25 mL aquiferous ethanol/g Folium Myricae rubrae with aquiferous ethanol, at the temperature of 60-90 ℃, adopt microwave auxiliary extraction method to extract 5-11 min, obtain Folium Myricae rubrae total flavones extracting solution, described aquiferous ethanol volumetric concentration is 60%-80%.
Concrete grammar of the present invention is: fresh Folium Myricae rubrae is dried, and pulverizes, and crosses 100 mesh sieves, with 10 times of volume petroleum ether, soak, and defat, sucking filtration, dries in the shade; Accurately weighed microwave extracting tank, proportionally add aquiferous ethanol and Folium Myricae rubrae, mix, weighed weight adopts microwave auxiliary extraction method to extract 5-11 min at the temperature of 60-90 ℃, standing cool to room temperature after extracting, weighed weight again, with extraction solvent, supply the weight of less loss, centrifugal 5 min under 3500 r/min, get supernatant and obtain Folium Myricae rubrae total flavones extracting solution.
As Folium Myricae rubrae total flavone extracting process of the present invention preferably: described aquiferous ethanol volumetric concentration is 70%-80%, Folium Myricae rubrae used and aquiferous ethanol ratio are 15-20 mL aquiferous ethanol/g Folium Myricae rubrae, temperature is 70-80 ℃, extraction time 7.00-9.00 min.
The present invention also provides a kind of simultaneously and take antioxidant activity as index, optimizes the method for Folium Myricae rubrae total flavone extracting process by Response Surface Method, and following 3 variablees are set: the liquid ratio of ethanol (80%) and Folium Myricae rubrae, extract temperature, extraction time; Utilize Design Expert software to be optimized according to Box-Behnken design principle.
By experiment of single factor, screen to choose and extract temperature, extraction time and three conditions of liquid ratio, carry out Box-Behnken experiment, respectively with X
1, X
2, X
3represent, the basic, normal, high test level code of each independent variable is respectively-1,0,1, in Table 1;
According to Box-Behnken design principle, carry out experimental program design, and test;
Utilize Design Expert software analysis experimental result to obtain the multinomial model of secondary of Folium Myricae rubrae total flavones antioxidant activity, filter out Folium Myricae rubrae total flavones optimum extraction condition.
The invention provides and a kind ofly take antioxidant activity as index, extract the optimization method of Folium Myricae rubrae total flavones, to extracting temperature, extraction time, liquid ratio is optimized the influence factor of Folium Myricae rubrae total flavones, the optimum optimizing condition obtaining is: extract 73.3 ℃ of temperature, extraction times 7.00 min, liquid ratio 20:1 mL/g.With DPPH (2,2-di-phenyl-1-picrylhydrazyl) method and ABTS (2,2 '-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt) in method mensuration extracting solution, the equivalent of Trolox (watermiscible vitamin E) is respectively 1352.10 μ mol/g and 1421.46 μ mol/g, and is 70.94 mg/g with the general flavone content of the AlCl3 method mensuration extract of improveing.
The present invention be take antioxidant activity as index, and by measuring by DPPH method and ABTS method, application responds Surface Method is optimized a kind of method of Folium Myricae rubrae total flavone extracting process.Only by 17 groups of experiments, can extract to the extraction conditions of Folium Myricae rubrae total flavones temperature, extraction time and these influence factors' of liquid ratio optimization, obtain optimum optimizing condition, and optimum yields, and this productive rate and antioxidant activity link together, to seeking natural inhibitor, be of practical significance.
The present invention is only by a small amount of experiment, just can optimize rationally and effectively to take the extraction process of the Folium Myricae rubrae total flavones that antioxidant activity is index, simple and feasible.
The present invention has good use value for Folium Myricae rubrae total flavones and antioxidant activity research.
Tool of the present invention has the following advantages and efficiency:
Take antioxidant activity as index, and application responds Surface Analysis method is optimized a kind of method of Folium Myricae rubrae total flavone extracting process.Only by 17 groups of experiments, just can be optimized the extraction process of Folium Myricae rubrae total flavones.
When extracting temperature, it is 73.3 ℃, extraction time is 7.00 min, when liquid ratio is 20:1 mL/g, the equivalent of measuring Trolox in extracting solution by DPPH method and ABTS method is respectively 1352.10 μ mol/g and 1438.73 μ mol/g, and the general flavone content of extract is 70.94 mg/g.The present invention has shortened extraction time, has improved extraction ratio; Reduce liquid ratio, reduced the consumption of organic solvent; Reduce extraction temperature, saved the energy.
In sum, it is response Surface Analysis method that Box-Behnken Design (BBD) center combination that the present invention adopts designs a model, with 3 changed factors, 3 levels, with a small amount of experimental group, just draw experimental result, the optimum condition of gained is not the value set but in the scope imposing a condition.Take antioxidant activity as index, by Response Surface Method, optimize the extraction conditions of Folium Myricae rubrae total flavones, and by being optimized extracting temperature, extraction time and these influence factors of liquid ratio, obtain optimum optimizing condition.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail.
Fig. 1 is for extracting temperature and the extraction time response surface graphics on the antioxidant activity impact of Waxberry leaf extract scavenging ability of DPPH free radical;
Fig. 2 is for extracting temperature and the solid-liquid ratio response surface graphics on the antioxidant activity impact of Waxberry leaf extract scavenging ability of DPPH free radical;
Fig. 3 is response surface graphics liquid ratio and extraction time the antioxidant activity of Waxberry leaf extract scavenging ability of DPPH free radical being affected;
Fig. 4 removes the response surface graphics of the antioxidant activity impact of ABTS free radical ability for extracting temperature and extraction time on Waxberry leaf extract;
Fig. 5 removes the response surface graphics of the antioxidant activity impact of ABTS free radical ability for extracting temperature and solid-liquid ratio on Waxberry leaf extract;
Fig. 6 removes the response surface graphics of the antioxidant activity impact of ABTS free radical ability at liquid ratio and extraction time on Waxberry leaf extract.
The specific embodiment
Take antioxidant activity as index, by Response Surface Method, optimize the method for Folium Myricae rubrae total flavone extracting process:
One, experimental implementation part:
The preparation of Trolox (watermiscible vitamin E) standard reserving solution: take a certain amount of Trolox, be placed in the volumetric flask of 10 ml, dilute and be settled to scale with anhydrous alcohol solution, shake up, obtain the Trolox standard reserving solution of 1 mg/ml.
The assay method of oxidation resistance and the making of standard curve:
The making of DPPH method Plays curve: Trolox standard reserving solution is diluted to 500 μ g/ml, by pipet precision, measure 1 ml, 0.8 ml, 0.6 ml, 0.4 ml, 0.2 ml, 0.0 ml Trolox solution is placed in 10 ml volumetric flasks, with anhydrous alcohol solution, dilutes and is settled to scale, shakes up, obtain the Trolox solution of variable concentrations, by DPPH method, measure its absorbance.
The making of ABTS method Plays curve: measure 0.55 ml by pipet precision, 0.5 ml, 0.45 ml, 0.4 ml, 0.35 ml, 0.3 ml Trolox standard reserving solution is placed in 10 ml volumetric flasks, with anhydrous alcohol solution, dilute and be settled to scale, shake up, obtain the Trolox solution of variable concentrations, by ABTS method, measure its absorbance.
The pre-treatment of Folium Myricae rubrae: fresh Folium Myricae rubrae is dried, pulverizes, and crosses 100 mesh sieves, with 10 times of volume petroleum ether, soak, and defat, sucking filtration, dries in the shade, as sample powder.
Weigh sample thief 0.70 g in the balance, accurately weighed microwave extracting tank, by liquid ratio, add ethanol (80%), weighed weight is extracted by the regular hour under different temperatures, standing cool to room temperature after extracting, weighed weight again, supplies the weight of less loss, centrifugal 5 min under 3500 r/min with extraction solvent, get supernatant, obtain extract.Dilute certain multiple, by DPPH method and ABTS method, measure the oxidation resistance of Folium Myricae rubrae total flavones, and represent with the equivalent of Trolox, and with the AlCl improveing
3method is measured the general flavone content of extract.
Two, experimental design and statistical analysis
1. experiment of single factor
Change solvent, percentage of solvents, extraction temperature, extraction time and liquid ratio and carry out experiment of single factor, by DPPH method and ABTS method, measure the oxidation resistance of Folium Myricae rubrae total flavones, and represent with the equivalent of Trolox, and with the AlCl improveing
3method is measured the general flavone content of extract.Each processing in triplicate.
2. Response Surface Method optimal design
According to single factor experiment result, choose the impact of total flavones extraction effect and extract more significantly temperature, extraction time and three factors of liquid ratio, utilize Design Expert software according to Box-Behnken design principle contrived experiment, choose extraction temperature, extraction time and liquid ratio and carry out the setting of empirical factor and level, set and the results are shown in table 1 in summary of the invention.
To extract temperature X
1, extraction time X
2with liquid ratio X
3for independent variable, the antioxidant activity (representing with the equivalent of Trolox) of total flavones of take is response value Y, EXPERIMENTAL DESIGN scheme and the results are shown in Table 2.
3. the foundation of model and statistical analysis
According to the data obtained, carry out multiple regression analysis, the polynary quadratic regression equation between the variable that meets with a response (extracting temperature, extraction time and liquid ratio) and response value (antioxidant activity of total flavones (representing with the equivalent of Trolox)).
Y
1(DPPH)=?1165.7?-?54.01X
1?-?33.21X
2?+?136.65X
3?+?9.71X
1X
2?-?36.75X
1X
3?-?21.98X2X
3?-?74.86X
1 2?-?30.04X
2 2?-?37.50X
3 2
Y
2(ABTS)=?1284.66?+?4.23X
1?–?40.94X
2+?153.73X
3?+?8.06X
1X
2?+?0.96X
1X
3?-?33.94X
2X
3?–?80.96X
1 2?–?43.05X
2 2?–?30.35X
3 2
Linear relationship significance between each factor and response value, is judged by the check of F value, P value is less, and the significance of explanatory variable is higher.From the analysis of variance table (table 3) of DPPH method and the analysis of variance table (table 4) of ABTS method, the linear relationship between its dependent variable and all independent variables is (R significantly
2be respectively 0.9823 and 0.9801), the significance level of model is <0.0001 all, so regression equation model is significant.
4. test result analysis and optimization
Utilize Design Expert software according to the regression equation analysis of drawing, obtain response surface and the equal pitch contour of regression equation, as depicted in figs. 1 and 2.
Known according to model analysis, the Extraction technique of the Folium Myricae rubrae total flavones that the antioxidant activity of take is index is DPPH method, extracts 73.3 ℃ of temperature, extraction times 7.00 min, liquid ratio 20:1 mL/g; ABTS method, 79.9 ℃ of temperature, times 7.00 min, liquid ratio 20:1 mL/g;
In triplicate, the equivalent of the Trolox of checking DPPH method and ABTS method is 1352.10 μ mol/g ± 15.83% in test, 1438.74 μ mol/g ± 9.00%, and with predictive value 1323.70 μ mol/g, 1440.62 μ mol/g approach.Fully verified the reasonability of institute's established model.
Consider the consumption that reduces costs and reduce the energy, last selective extraction technological parameter is: extract 73.3 ℃ of temperature, extraction times 7.00 min, liquid ratio 20:1 mL/g.With this understanding, the equivalent of the Trolox of DPPH method and ABTS method is 1352.10 μ mol/g ± 15.83%, 1421.46 μ mol/g ± 9.50%, the AlCl of improvement
3the content that method is measured total flavones is 70.94 mg/g ± 0.308% (n=3).
Embodiment 1.
Fresh Folium Myricae rubrae is dried, and pulverizes, and crosses 100 mesh sieves, with 10 times of volume petroleum ether, soak, and defat, sucking filtration, dries in the shade, as sample powder; By 0.7 g sample powder and 14 mL ethanol (80%) (that is, liquid ratio 20:1), microwave auxiliary extraction 7.00 min at the temperature of 73.3 ℃, under 3500 r/min, centrifugal 5 min, get supernatant, obtain extract.Dilute certain multiple, gained extract is surveyed antioxidant activity by DPPH method and ABTS method, then with the AlCl improveing
3method records the content of total flavones, and the content of the total flavones obtaining is 70.94 mg/g.
Embodiment 2.
Fresh Folium Myricae rubrae is dried, and pulverizes, and crosses 100 mesh sieves, with 10 times of volume petroleum ether, soak, and defat, sucking filtration, dries in the shade, as sample powder; By 0.7 g sample powder and 10.5 mL ethanol (80%) (that is, liquid ratio 15:1), microwave auxiliary extraction 7.00 min at the temperature of 60 ℃, under 3500 r/min, centrifugal 5 min, get supernatant, obtain extract.Dilute certain multiple, gained extract is surveyed antioxidant activity by DPPH method and ABTS method, then with the AlCl improveing
3method records the content of total flavones, and the content of the total flavones obtaining is 65.41 mg/g.
Embodiment 3.
Fresh Folium Myricae rubrae is dried, and pulverizes, and crosses 100 mesh sieves, with 10 times of volume petroleum ether, soak, and defat, sucking filtration, dries in the shade, as sample powder; By 0.7 g sample powder and 7 mL ethanol (80%) (that is, liquid ratio 10:1), microwave auxiliary extraction 9.00 min at the temperature of 80 ℃, under 3500 r/min, centrifugal 5 min, get supernatant, obtain extract.Dilute certain multiple, gained extract is surveyed antioxidant activity by DPPH method and ABTS method, then with the AlCl improveing
3method records the content of total flavones, and the content of the total flavones obtaining is 64.06 mg/g.
Embodiment 4.
Fresh Folium Myricae rubrae is dried, and pulverizes, and crosses 100 mesh sieves, with 10 times of volume petroleum ether, soak, and defat, sucking filtration, dries in the shade, as sample powder; By 0.7 g sample powder and 14 mL ethanol (80%) (that is, liquid ratio 20:1), microwave auxiliary extraction 5.00 min at the temperature of 70 ℃, under 3500 r/min, centrifugal 5 min, get supernatant, obtain extract.Dilute certain multiple, gained extract is surveyed antioxidant activity by DPPH method and ABTS method, then with the AlCl improveing
3method records the content of total flavones, and the content of the total flavones obtaining is 68.09 mg/g.
Embodiment 5.
Fresh Folium Myricae rubrae is dried, and pulverizes, and crosses 100 mesh sieves, with 10 times of volume petroleum ether, soak, and defat, sucking filtration, dries in the shade, as sample powder; By 0.7 g sample powder and 17.5 mL ethanol (80%) (that is, liquid ratio 25:1), microwave auxiliary extraction 11.00 min at the temperature of 90 ℃, under 3500 r/min, centrifugal 5 min, get supernatant, obtain extract.Dilute certain multiple, gained extract is surveyed antioxidant activity by DPPH method and ABTS method, then with the AlCl improveing
3method records the content of total flavones, and the content of the total flavones obtaining is 63.15 mg/g.
Embodiment 6.
Fresh Folium Myricae rubrae is dried, and pulverizes, and crosses 100 mesh sieves, with 10 times of volume petroleum ether, soak, and defat, sucking filtration, dries in the shade, as sample powder; By 0.7 g sample powder and 17.5 mL ethanol (60%) (that is, liquid ratio 25:1), microwave auxiliary extraction 40.00 min at the temperature of 70 ℃, extract 2 times, and under 3500 r/min, centrifugal 5 min, get supernatant, obtain extract.Dilute certain multiple, gained extract is surveyed antioxidant activity by DPPH method and ABTS method, then with the AlCl improveing
3method records the content of total flavones, and the content of the total flavones obtaining is 32.11 mg/g.
Visible, the content of total flavones is very low, may be because extraction time is oversize, the broken structure of having encircled target compound.
And existing extractive technique, with the extracting method of embodiment 6, with ultrasonic-microwave synergistic extraction Folium Myricae rubrae total flavones, extraction times 40 min, extracts 2 times, and the extraction ratio of Folium Myricae rubrae total flavones is 2.28%.
What more than enumerate is only several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, can also have a lot of distortion.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention, all should think protection scope of the present invention.
Claims (8)
1. the extraction process of Folium Myricae rubrae total flavones, it is characterized in that: Folium Myricae rubrae mixes according to the liquid ratio of 10-25 mL aquiferous ethanol/g Folium Myricae rubrae with aquiferous ethanol, at the temperature of 60-90 ℃, adopt microwave auxiliary extraction method to extract 5-11 min, obtain Folium Myricae rubrae total flavones extracting solution, described aquiferous ethanol volumetric concentration is 60%-80%.
2. the extraction process of Folium Myricae rubrae total flavones according to claim 1, is characterized in that: fresh Folium Myricae rubrae is dried, and pulverizes, and sieves, with petroleum ether, soak, and defat, sucking filtration, dries in the shade; Accurately weighed microwave extracting tank, according to liquid ratio, mix, weighed weight, at the temperature of 60-90 ℃, adopt microwave auxiliary extraction method to extract 5-11 min, extraction is standing cool to room temperature afterwards, more weighed weight, supplies the weight of less loss with extraction solvent, centrifugal 5 min, get supernatant and obtain Folium Myricae rubrae total flavones extracting solution.
3. extraction process according to claim 1 and 2, is characterized in that: described aquiferous ethanol concentration is 70%-80%.
4. extraction process according to claim 1 and 2, is characterized in that: described temperature is 70-80 ℃.
5. extraction process according to claim 1 and 2, is characterized in that: extraction time is 7-9 min.
6. the extraction process of Folium Myricae rubrae total flavones according to claim 1, is characterized in that: described Folium Myricae rubrae mixes according to the liquid ratio of 15-20 mL aquiferous ethanol/g Folium Myricae rubrae with aquiferous ethanol.
7. the application of Response Surface Method in optimizing extracting technique of Chinese medicine.
8. application according to claim 7, is characterized in that, described Chinese medicine is Folium Myricae rubrae, and following 3 variablees are set: the liquid ratio of Folium Myricae rubrae and aquiferous ethanol, extraction temperature, extraction time; Utilize Design Expert software to be optimized according to Box-Behnken design principle.
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| CN104782959A (en) * | 2015-04-13 | 2015-07-22 | 邵素英 | Feed for promoting development of ovary and fallopian tube of laying hen and preparation method of feed |
| CN104839466A (en) * | 2015-04-13 | 2015-08-19 | 邵素英 | Laying chicken reproductive performance improving material in the egg laying peak prophase and preparation method thereof |
| CN107259292A (en) * | 2017-03-24 | 2017-10-20 | 浙江省农业科学院 | Polyphenoils is extracted and method for preserving in a kind of Waxberry fruit |
| CN107132290A (en) * | 2017-05-16 | 2017-09-05 | 云南农业大学 | A kind of method that Pu'er cooked tea flavone compound is extracted based on response phase method |
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